Indian Patents. 202293:ISOLATION OF INNER CELL MASS FOR THE ESTABLISHMENT OF HUMAN EMBRYONIC STEM CELL (HESC) LINES

Title of Invention	ISOLATION OF INNER CELL MASS FOR THE ESTABLISHMENT OF HUMAN EMBRYONIC STEM CELL (HESC) LINES
Abstract	. A method for establishing cell lines from the inner cell mass of a human blastocyst, comprising the steps of: (a) isolating a blastocyst; comprises zona pellucida, trophectoderm and inner cell mass. (b) creating an aperture in the blastocyst by laser ablation; and (c) Isolating cells of the inner cell mass from the blastocyst through the aperture.

Full Text	FORM 2 THE PATENTS ACT, 1970 (39 of 1970) COMPLETE SPECIFICATION SECTION 10 " ISOLATION OF INNER CELL MASS FOR THE ESTABLISHMENT OF HUMAN EMBRYONIC STEM CELL (hESC) LINES " RELIANCE LIFE SCIENCES PVT.LTD. an Indian Company having its Registered office at Chitrakoot, 2nd Floor, Shree Ram Mills Compound, Ganpath Rao Kadam Marg, Worli, Mumbai -400 013, Maharashtra, India. The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:- GRANTED ORIGINAL 107/MUM/2003 Title Isolation of inner cell mass for the establishment of human embryonic stem cell (hESC) lines. Related Application: This application claims priority to the PCT application No PCT/IN02/00168 dated August 20, 2002. Field of the Invention: The present invention relates to a method of isolation of inner cell mass (ICM) derived from embryo for establishing human embryonic stem cell (hESC) lines, using a non-contact diode laser technique. Background of the invention The isolation of human stem cells offers the promise of a remarkable array of novel therapeutics. Biologic therapies derived from such cells through tissue regeneration and repairs as well as through targeted delivery of genetic material are expected to be effective in the treatment of a wide range of medical conditions. Efforts to analyze and assess the safety of using human stem cells in the clinical setting are vitally important to this endeavor. Embryonic stem (ES) cells are the special kind of cells that can both duplicate themselves (self renew) and produce differentiated functionally specialized cell types. These stem cells are capable of becoming almost all of the specialized cells of the body and thus, may have the potential to generate replacement cells for a broad array of tissues and organs such as heart, pancreas, nervous tissue, muscle, cartilage and the like. Stem cells have the capacity to divide and proliferate indefinitely in culture. Scientists use these two properties of stem cells to produce seemingly limitless supplies of most human cell types from stem cells, paving the way for the treatment of diseases by cell replacement. In fact, cell therapy has the potential to treat any disease that is associated with cell dysfunction or damage including stroke, diabetes, heart attack, spinal cord injury, cancer and AIDS. The potential of manipulation of stem cells to repair or replace diseased or damaged tissue has generated a great deal of excitement in the scientific, medical and/ biotechnology investment communities. ES cells from various mammalian embryos have been successfully grown in the laboratory. Evans and Kaufman (1981) and Martin (1981) showed that it is possible to derive permanent lines of embryonic cells directly from mouse blastocysts. Thomson et al., (1995 and 1996) successfully derived permanent cell lines from rhesus and marmoset monkeys. Pluripotent cell lines have also been derived from pre-implantation embryos of several domestic and laboratory animal species such as bovines (Evans et al., 1990) Porcine (Evans et al., 1990, Notarianni et al., 1990), Sheep and goat (Meinecke-Tillmann and Meinecke, 1996, Notarianni et al., 1991), rabbit (Giles et al.„, 1993, Graves et al., 1993) Mink (Sukoyan et al., 1992) rat (lannaccona et al., 1994) and Hamster (Doetschman et al., 1988). Recently, Thomson et al (1998) and Reubinoff et al (2000) have reported the derivation of human ES cell lines. These human ES cells resemble the rhesus monkey ES cell lines. ES cells are found in the ICM of the human blastocyst, an early stage of the developing embryo lasting from the 4th to 7th day after fertilization. The blastocyst is the stage of embryonic development prior to implantation that contains two types of cells viz. 1. Trophectoderm: outer layer which gives extra embryonic membranes. 2. Inner cell mass (ICM): which forms the embryo proper. In normal embryonic development, ES cells disappear after the 7th day and begin to form the three embryonic tissue layers. ES cells extracted from the ICM during the blastocyst stage, however, can be cultured in the laboratory and under the right conditions proliferate indefinitely. ES cells growing in this undifferentiated state retain the potential to differentiate into cells of all three embryonic tissue layers. Ultimately, the cells of the inner cell mass give rise to all the embryonic tissues. It is at this stage of embryogenesis, near the end of first week of development, that ES cells can be derived from the ICM of the blastocyst. The ability to isolate ES cells from blastocysts and grow them in culture seems to depend in large part on the integrity and condition of the blastocyst from which the cells are derived. In short, the blastocyst that is large and has distinct inner cell mass tend to yield ES cells most efficiently. Several methods have been used for isolation of inner cell mass (ICM) for the establishment of embryonic stem cell lines. Most common methods are as follows: Natural hatching of the blastocyst: In this procedure blastocyst is allowed to hatch naturally after plating on the feeder layer. The hatching of the blastocyst usually takes place on day 6. The inner cell mass (ICM) of the hatched blastocyst develops an outgrowth. This outgrowth is removed mechanically and is subsequently grown for establishing embryonic stem cell lines. However, this procedure has few disadvantages. Firstly, Trophectoderm cells proliferate very fast in the given culture conditions and many a times, suppress the outgrowth of inner cell mass. Secondly, while removing the outgrowth of the inner cell mass mechanically, there is a chance of isolating trophectoderm cells. Thirdly, the percentage of blastocysts hatching spontaneously in humans is very low. Microsurgery: Another method of isolation of inner cell mass is mechanical aspiration called microsurgery. In this process, the blastocyst is held by the holding pipette using micromanipulator system and positioned in such a way that the inner cells mass (ICM) is at 9 O'Clock position. The inner cell mass (ICM) is aspirated using a biopsy needle which is beveled shape and is inserted into the blastocoel cavity. This procedure too is disadvantageous as the possibility to isolate the complete inner cell mass is low and many a time cells get disintegrated. It is a very tedious procedure and may cause severe damage to the embryo. The operation at the cellular level requires tools with micrometer precision, thereby minimizing damage and contamination. Immunosurgery: This is a commonly used procedure to isolate inner cell mass (ICM). The inner cell mass (ICM) is isolated by complement mediated lysis. In this procedure, the blastocyst is exposed either to acid tyrode solution or pronase enzyme solution in order to remove the zona pellucida (shell) of blastocyst. The zona free embryo is then exposed to human surface antibody for about 30 min to one hour. This is followed by exposure of embryos to guinea pig complement in order to lyse the frophectoderm. This complement mediated lysed trophectoderm cells are removed from inner cell mass (ICM) by repeated mechanical pipetting with a finely drawn Pasteur pipette. All the embryonic stem cell lines reported currently in the literature have been derived by this method. However, this method has several disadvantages. Firstly, the embryo is exposed for a long time to acid tyrode or pronase causing deleterious effects on embryo, thereby reducing the viability of embryos proper. Secondly, it is time consuming procedure as it takes about 1.5 to 2.0 hours. ( Narula et al.,1996). Thirdly, the yield of inner cell mass (ICM) per blastocyst is low. Fourthly, critical storage conditions are required for antibody and complement used in the process. Lastly, it involves the risk of transmission of virus and bacteria of animal origin to humans, as animal derived antibodies and complement are used in the process. In this process, two animal sera are used. One is rabbit antihuman antiserum and the other is guinea pig complement sera. The human cell lines studied to date are mainly derived by using a method of immunosurgery, where animal based antisera and complement was used. Other possible disadvantages of the existing cell lines are as follows:- 1 Use of feeder cells for culturing the human embryonic stem cell (hESC) lines produces mixed cell population that require the Embryonic stem cells (ESC) to be separated from feeder cell components and this impairs scale up. 2. Embryonic stem cells (ESC) get contaminated by transcripts from feeder cells and cannot be used on a commercial scale. It can be used only for research purposes. Geron established a procedure where human Embryonic Stem Cell (hESC) line was cultured in the absence of feeder cells (Xu et.al., 2001). The hESC were cultured on an extracellular matrix in a conditioned medium and expanded in this growth environment in an undifferentiated state. The hESC contained no xenogenic components of cancerous origin from other cells in the culture. Also, the production of hESC cells and their derivatives were more suited for commercial production. In this process, there was no need to produce feeder cells on an ongoing basis to support the culture, and the passaging of cells could be done mechanically. However, the main disadvantage of this procedure is that the inner cell mass (ICM) is isolated by immunosurgery method, wherein the initial derivation of Embryonic Stem Cells is carried out using feeder layer containing xenogenic components. This raises the issue of possible contamination with animal origin viruses and bacteria. In order to simplify the procedure of inner cell mass isolation and to make it safe, the scientists of the present invention have come out with a novel method of isolation of the inner cell mass using a non-contact laser, wherein, the use of animal based antisera and complement have been eliminated. Use of Laser technique in Assisted Reproduction: With the advent of assisted reproductive technologies (ART), several methods have been used for improving fertilization, facilitating blastocyst hatching (Cohen et al, 1990) and performing blastomere biopsy (Tarin and Handyside, 1993). Commonly used methods are chemical (Gordon and Talansky, 1986), mechanical (Depypere et al., 1988) and laser (Feichtinger et al., 1992) so as to produce holes in the zona pellucida (Gordon, 1988). Recently, an infrared 1.48 pm diode laser beam focused through a microscope objective was shown to allow rapid, easy and non-touch microdrilling of mouse and human zona pellucida and high degree of accuracy was maintained under conventional culture conditions (Rink et al., 1994). The drilling effect was shown due to a highly localized heat-dependent disruption of the zona pellucida glycoprotein matrix (Rink et al., 1996). Contrary to the detrimental effect on compacted mouse embryos induced by the 308 nm xenon-chlorine excimer laser (Neev et al., 1993), the drilling process in the infrared region did not affect embryo survival in mice (Germond et al., 1995) or in humans (Antinori et al., 1994). Currently, laser is being investigated as a tool to aid fertilization and in assisted hatching. Recent reports show that use of 1.48 pm diode laser for microdrilling mouse zona pellucida is highly safe and does not affect neuro-anatomical and neurochemical properties in mice and also improves fertilization (Germond et al., 1996). Obruca and colleagues first reported the success of laser-assisted hatching in human IVF in 1994. In this study, a 20- to 30-micron hole was made in the zona pellucida ( ZP ) when the embryos were at the two- to four-cell stage, and embryos were transferred immediately. Patients with previous IVF failures from two separate centers were included in this study. There was a higher implantation rate per embryo in the laser-assisted hatching group (14.4%) versus the control group (6%). Pregnancy rates per transfer were also improved (40% versus 16.2%). In a separate study, Er:YAG laser was used to thin the ZP of embryos derived from patients undergoing repeated IVF. Using a laser for thinning the ZP, embryologists are able to achieve accurate reduction of the ZP by 50%, which is very difficult with acidic Tyrode's solution. Presence of Acid Tyrode's solution near the embryo may also be detrimental. The rate of clinical pregnancies in the laser-hatched group was 42.7%, as compared to 23.1% in the control unhatched group. Since this data looked promising, the indication of laser-assisted hatching was extended. Women undergoing IVF for the first time yielded 39.6% clinical pregnancy rate in the laser-treated group versus a 19% rate in the control unhatched group (Parikh et al 1996). During the last decade there has been ongoing research on the isolation of inner cell mass (ICM), as it is useful in establishing embryonic stem cell lines which in turn have the ability to develop into most of the specialized cells in the human body including blood, skin, muscle and nerve cells. They also have the capacity to divide and proliferate indefinitely in culture. The present invention involves the isolation of inner cell mass (ICM), using laser ablation technique without undergoing the cumbersome procedure of immunosurgery. Hence, in the present invention, the use of animal derived antibodies or sera are eliminated and the procedure is safe, simple, rapid and commercially viable. The present invention, obviates the shortcomings associated with the conventional methods of isolation of inner cell mass (ICM). The inner cell mass (ICM) isolated by the present invention is found to be intact without causing any destruction or damage to the cells. The present invention thus provides a quick reliable and non-invasive method for isolation of inner cell mass (ICM). It also completely ruptures the trophectoderm thereby minimizing the contamination of inner cell mass (ICM), thus ensuring the purity of inner cell mass (ICM). REFERENCES 1. Antinori S, Versaci C, Fuhrberg P et al (1994). Seventeen live birth after the use of erbium-yytrium aluminum garnet laser in the treatment of male factor infertility. Hum Reprod. 9 : 1891-1896. 2. Cohen J, Eisner C, Kort H et al (1990). Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod . 5 : 7-13 3. Depypere HT, McLaughlin KJ, Seamark RF et al (1988). Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse. J. Reprod Fertil. 84:205-211. 4. Doetschman T, Williams P and Maeda N (1988) Establishment of hamster blastocyst derived embryonic stem (ES) cell. Developmental Biology 127: 224-227. 5. Evans MJ and Kaufman MH (1981). Establishment in culture of pluripotential cells from mouse embryo. Nature 292: 154-156. 6. Evans MJ, Notarianni E, Laurie S and Moor RM (1990) Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocyst. Theriogenology 33:125-128. 7. Feichtinger W, Strohmer H, Fuhrberg P et al (1992). Photoablation of oocyte zona pellucida by erbium-yag laser for in-vitro fertilization in severe male infertility.Lancet. 339, 811. 8. Gordon JW (1988). Use of micromanipulation for increasing the efficiency of mammalian fertilization in vitro. Ann. N.Y. Acad.Sci. 541 : 601-613. 9. Gordon JW and Talansky BE (1986). Assisted fertilization by zona drilling : a mouse model for correction of oligospermia. J. Exp Zool. 239: 347-354. 10. Germond M, Nocera D, Senn A. Rink K. et al (1995). Microdissection of mouse and human zona pellucida using 1.48 microns diode laser beam: efficacy and safety of the procedure. Fertil Steril. 64: 604-611. 11. Germond M, Nocera D, Senn A, Rink A et al (1996). Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 micron diode laser beam. Hum Reprod. 11: 1043-1048 12. Giles JR, Yang X, Mark X and Foot RH (1993). Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetus following injection into blastocysts or morula. Molecular Reproduction and Development 36: 130-138. 13. Graves KH and Moreadith RW (1993). Derivation and characterization of putative pluripotential embryonic stem cells from pre-implantation rabbit embryo. Molecular reproduction and Development 36: 424-433. 14. lannaccone PM, faborn GU, Garton RL et al (1994). Pluripotent embryonic stem cells from the rat are capable of producing chimeras. Developmental Biology 163:288-292. 15. Martin GR (1981) Isolation of pluripotent cell lines from early mouse embryos cultured in medium conditioned with teratocarcinoma stem cells. Proceeding of National Academy of Sciences USA 72: 1441-1445. 16. Meinecke-Tillmann S and Meinecke B (1996). Isolation of ES like cell lines from ovine and caprine pre-implantation embryo. J Animal Breeding and Genetics 113:413-426. 17. Narula A, Taneja, Totey SM (1996) Morphological cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryo: the effect of IGF-I. Mol. Reprod. Dev. 44(3):343-51.. 18. Neev J, Gonzales A, Lucciardi F et al (1993). Opening of the mouse zona pellucida by laser without a micromanipulator. Hum Reprod . 8 : 939-944. 19. Obruca A, Strohmer H, Sakkas D (1994). Use of laser in assisted fertilization and hatching. Hum Reprod. 9:1723-1726. 20. Parikh FR, Kamat SA, Nadkarni S et al (1996). Assisted hatching in an in vitro fertilization program. J Reprod Fertil Suppl 50:121-125. 21. Reubinoff BE, Per MF, Fong CY, Trounson A and Bongso A (2000) Embryonic stem cell lines from human blastocysts: Somatic differentiation in vivo. Nat Biotechnol. 18:299-304. 22. Rink K, Delacretaz G, Salathe RP et al (1994). Proceedings SPIE. 2134A, 412-422. 23. Rink K, Delacretaz G, Salathe RP et al (1996). Non-contact microdrilling of mouse zona pellucida with an objective delivered 1.48 microns diode laser. Lasers Surg Med. 18:52-62. 24. Sukoyan MA, Golublitsa AN, Zhelezova Al et al (1992) Isolation and cultivation of blastocyst derived stem cell lines from American Mink. Molecular Reproduction and Development 33: 418-431. 25. Tarin JJ and Handyside AH (1993). Embryo biopsy strategies for preimplantation diagnosis. Fertil Steril 59:943-952. 26. Thomson JA, Itskovitz-Eldor J, Shapiro SS. et al. (1998). Embryonic stem cell lines derived from human blastocyst. Science 282 : 1145-1147. 27. Thomson JA, Kalishman J, Golos TG et al (1996). Pluripotent cell line derived from common marmoset blastocyst. Biology of Reproduction. 55: 254-259 Objects of the Invention 1. It is an object of the present invention, to develop a process of isolation of inner cell mass, using laser ablation technique, without undergoing the cumbersome procedure of immunosurgery. 2. It is another object of the present invention to isolate ICM using laser ablation technique without using any animal generated antibodies and sera, thereby preventing the possibility of transmission of animal organism to human and thus can safely be used on commercial scale. 3. It is another object of the present invention to isolate inner cell mass (ICM) from blastocyst stage of a mammalian embryo using a non-contact diode laser. 4. It is another object of the present invention to isolate inner cell mass (ICM) by simple, shorter and easily feasible way without affecting/destroying the inner cell mass (ICM). 5. It is still another object of the present invention to ensure the purity of inner cell mass (ICM) by ablating completely trophectoderm thereby minimising the contamination of inner cell mass (ICM). 6. It is still another object of the present invention to isolate inner cell mass (ICM) of high yield and purity as compared to the inner cell mass (ICM) isolated by the conventional methods. These and other objects of the invention will become more readily apparent from the ensuing description. Details of Invention: The present invention relates to isolation of inner cell mass, using laser ablation of zona pellucida (ZP) and trophectoderm (TE) and aspiration of inner cell mass for establishing embryonic stem cell lines. In the present invention, the non contact diode laser used is highly accurate and reliable tool for cellular microsurgery. The system incorporates the latest in fiber optic technology to provide the most compact laser system currently available. The 1.48 pm diode non-contact Laser System is mounted/implanted via the epifluorescence port to inverted microscope fitted with micromanipulators. A pilot laser is used to target the main ablation laser and a series of LEDs inform the user when the laser is primed and is ready to fire. A two-second-operation window is used to reduce the possibility of accidentally firing the laser. The spot diameter of the laser can be varied according to the hole size required. Couples undergoing in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) treatment voluntarily donate surplus human embryos. These embryos are used for research purposes after taking the informed written, voluntary consent from these couples. In the present invention, blastocyst stage embryos are taken for the isolation of inner cell mass. The blastocyst is placed in a petridish in a microdroplets of embryo biopsy medium with or without Ca++/Mg++ and is covered with mineral oil. The micromanipulator is set up to perform the embryo biopsy procedure. The blastocyst is placed in embryo biopsy medium and the petridish containing the blastocyst is placed on the heated stage of the microscope. The blastocyst is positioned at the center of the field. The blastocyst is immobilized on to the holding pipette in such a way that the inner cell mass is at 3 O' Clock position. The zona pellucida and trophectoderm close to inner cell mass is positioned on the aiming spot of the laser beam. A small portion of zona pellucida and trophectoderm is laser ablated. Biopsy pipette is then gently inserted through the hole in the zona pellucida and trophectoderm and the inner cell mass is gently aspirated. After isolation of the complete inner cell mass, the cells are given several washes with embryonic stem cell (ESC) medium. The cells are then plated on to coated plate in the presence of embryonic stem cell medium, with or without feeder layer for establishing embryonic stem cell lines. The embryonic stem cells were then characterized for cell surface markers such as SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4 and alkaline phosphatase. The embryonic stem cell lines are also karyotyped. a) Development of blastocyst in vitro: Institutional Ethics Committee approval has been obtained before initiation of this study. Prior written consent was taken from individual donor for the donation of surplus embryos for this study after completion of infertility treatment. Protocol generally used for infertility patients for obtaining viable embryo is as follows: The ovarian superovulation began with gnRH agonist analog suppression daily starting in the mid-luteal phase and administered in doses of 500-900 mgs for about 9-12 days. Ovarian stimulation was started after adequate ovarian suppression with human menopausal gonadotropins (hMG) or recombinant follicle stimulating hormone (FSH) (Gonal-F, Recagon) in appropriate doses depending on the age and ovarian volume. The dose was also adjusted as necessary to produce controlled ovarian stimulation. Serum beta-estradiol (E2) measurements were carried out as required. Transvaginal ultrasound was performed daily from cycle day 7 onward. Human Chorionic gonadotropin 5000-10000 I.U. was administered when three or more follicles were at least 17 mm in largest diameter. Transvaginal aspiration was performed 34-36 h later. Oocytes were then subjected to intracytoplasmic sperm injection. A holding pipette was used to secure the egg. Motile sperm were placed in a drop of polyvinyl pyrolidone (PVP) solution and overlaid with mineral oil. An injection needle was used to pierce the zona pellucida at about 3 O' Clock position. The selected spermatozoon was immobilized by cutting the tail with the injection micropipette. The oocyte was secured with holding pipette and spermatozoon was injected directly into the center of the oocyte. Oocytes were checked after 16-18 hours of culture for fertilization. At this point the fertilized oocyte had pro-nuclei (also called one cell embryo). One-cell embryos were then transferred into pre-equilibrated fresh ISM-1 medium or any other suitable embryo culture medium and incubated at 37 ° C in a 5% C02 in air. The next day embryos were transferred into ISM-2 medium or any oVneT suitabie embryo culture medium. Every alternate day embryos were transferred into fresh ISM-2 medium or any other suitable embryo culture medium. From day 5 onward embryos were checked for the blastocyst development. After the treatment is over, the surplus blastocysts were donated by the couples for this research work. b) Setting up of the Laser: The present invention relates to describing a unique method for inner cell mass isolation for establishment of embryonic stem cells using the non-contact diode laser. The laser is highly accurate and reliable tool for cellular microsurgery. The system incorporates the latest in fiber optic technology to provide the most compact laser system currently available. The 1.48 pm non-contact diode Laser System was mounted via the epifluorescence port to inverted microscope fitted with micromanipulators. A pilot laser was used to target the main ablation laser and a series of LED's inform the user when the laser is primed and ready to discharge the laser beam. A two-second-operation window was used to reduce the possibility of accidentally firing the laser. The spot diameter of the laser can be varied according to the ablation size required. c) Laser Ablation and isolation of inner cell mass. The blastocyst was individually placed in a microdroplet of biopsy medium (with or without Ca *7Mg ++ in a petri dish. The embryo was immobilized with the holding pipette in such a way that the inner cell mass remained at 3 O' Clock position and the zona pellucida and trophectoderm close to inner cell mass positioned on the aiming spot. A 1.48 urn diode laser was used to create an aperture in the Zona Pellucida (ZP), which is a glycoprotein layer protecting the oocyte/embryo. At this wavelength, the hole was induced by a local thermo-dissolution of the glycoprotein matrix. Once the zona pellucida was dissolved, trophectoderm cells were ablated by giving 3 or more pulses to cause photolysis. After ablation of both zona pellucida and trophectoderm, the aspiration pipette was introduced through laser-ablated hole and ICM was removed by gentle aspiration, with the aspiration pipette of required diameter. d) Culturing of human Embryonic Stem Cells (hESC) Prior to culturing, the aspirated ICM was washed thoroughly in ES medium. Given below is the procedure when the invention was carried out using feeder layer. In this process, the inner cell mass was cultured in tissue culture plate in the presence of inactivated mouse embryonic fibroblast feeder layer. Mouse embryonic fibroblast feeder layer was preferably obtained from Theiler stage mouse embryos i.e. 21 &22 (12.5 -15 days port coital) C57BL/6 mice or C57BL/6XSJL F-1 mice or out bred CD1 mice or from human amniotic fluid and used as a feeder layer. The fibroblast feeder layer was inactivated by gamma irradiation (3500 rads) or treatment with colchicine. The inactivated mouse embryonic fibroblast feeder layer was cultured on 0.5% gelatin coated plate with ES medium consisting of Dulbecco's modified Eagle's medium without Sodium pyruvate with high glucose contain (70-90%), Fetal bovine serum (10-30%), beta-mercaptoethanol, non-essential amino acids, L- Glutamine, basic fibroblast growth factor. After 4-7 days, ICM derived masses were removed from outgrowth with sterile fire polished pipette and were dissociated mechanically or with any other alternative method and plated on fresh feeder cells. Established cell lines were karyotyped and characterized for several surface markers such as SSEA-1, SSEA-3, SSEA-4, OCT-4, Alkaline phosphatase, TRA-1-81, TRA-1-60 as described by Thomson et al., (1998), Reubinoff et al., (2000). Examples The following examples are intended to illustrate the invention but do not limit the scope thereof. Example I: Total of 24 blastocyst stage human embryos were used for the isolation of inner cell mass. Embryos were washed several times in blastocyst culture medium (ISM-2 medium, Medicult, Denmark). Biopsy dish was prepared containing 50 \|il microdroplets of Ca++/ Mg++ free embryo biopsy medium (EB 10 medium, Scadinavian) overlaid with mineral oil and individual blastocyst was then placed in the microdroplet. Micromanipulator was set up. A holding pipette with outer diameter 75 jam and inner diameter 15 pm was used to secure the embryo. Biopsy pipette with an outer diameter of roughly 49 pm and inner diameter 35 prn was used for aspiration of inner cell mass. A pilot laser was used to target the main ablation laser. Blastocyst was immobilized with the holding pipette in such a way that inner cell mass remained at 3 O' clock position and the zona pellucida and trophectoderm close to inner cell mass positioned to the aiming spot. The hole was induced by a local thermo-dissolution of the zona. Trophectoderm cells were ablated by giving 3 pulses to cause photolysis. After ablation of both the zona pellucida and trophectoderm, the biopsy pipette was introduced through laser ablated hole and inner cell mass was removed. Inner cell mass was then washed several times in ES medium and placed in tissue culture plate in the presence or absence of feeder layer. The efficiency of the present invention i.e. isolation of inner cell mass by laser ablation method was compared with isolation of inner cell mass with conventional method i.e. immunosurgery. The comparative data is given below; Table 1: Summary of hESC lines developed using Laser ablation Technique of the present invention with the use of mouse feeder cells. No. of Total inner With mouse feeder cells blastocyst cell mass used for laser removed No- of No of ES cell lines established ablation ICM used 24 18 14 4 Example 2: Similarly, experiments were conducted with conventional method of isolation of inner cell mass i.e. using immunosurgery as given below: A total of 21 human blastocyst were used for isolation of inner cell mass. Embryos were washed several times with blastocyst culture medium (ISM-2 medium) and followed by ES medium. Individual blastocyst was then placed in 50 pl micodrops of 1:50 anti-human antibody (Sigma) for 30 minutes at 37 o C and 5% C02 in air. After incubation blastocyst were then washed four times with ES medium! Blastocysts were then placed in 50 (il of microdroplets of guinea pig complement at the concentration of 1:10 for 10 minutes at 37°C and 5% C02 in air. After incubation blastocyst were washed several times in ES medium using fine bore glass pipette in order to remove trophectoderm. Isolated inner . cell mass was then washed with ES medium and cultured in tissue culture plate in the presence or absence of feeder cells. Data are presented in the table: Table 2: Summary of hESC lines developed using immunosurgery with / without the use of mouse feeder cells. No. of Total With mouse feeder cells Without mouse feeder cells blastocyst Inner cell used for mass No. of No of ES cell No. of ICM used No. of ES laser removed ICM used lines cell lines ablation established established 21 14 .12 3 2 0 Although the isolation of inner cell mass using both the methods did not show any significant difference, the isolation of inner cell mass by laser ablation has distinct advantage. This method eliminates the use of antibodies and sera of animal origin. Isolation of inner cell mass by laser ablation method of the present invention can be cultured in the presence or absence of feeder layer. However, culturing of inner cell mass in a feeder free condition will further eliminate the possibilities of contamination of ES cell lines with animal viruses or bacteria and can be commercially utilized for human transplantation studies. In the current experiments, efforts were also made to establish ES cell line in the absence of feeder cells. Detailed description of the preferred embodiments: A preferred embodiment of the invention is illustrated in the accompanying photomicrographs. Fig 1 (a) to 1 (g) of the present invention, pertains to the isolation of inner cell mass (ICM) from the blastocyst of one embryo and Fig 2(a) to 2(g) pertains to the isolation of inner cell mass (ICM) from the blastocyst of another embryo. Fig 3,4,and 5 pertains to culturing of ICM on feeder cells at different stages. Fig 1. (a) shows a photomicrograph of human blastocyst, secured with glass holding pipette such that the ICM is at 3 O' Clock position. Fig 1 (b) shows a photomicrograph wherein part of zona pellucida and trophectoderm ablated with laser (arrow). Fig 1 (c) shows a photomicrograph of aspiration pipette close to the blastocyst following zona and trophectoderm ablation. Fig 1 (d) shows a photomicrograph of beginning of aspiration of ICM with aspiration pipette. Fig 1 (e) shows a photomicrograph of large portion of ICM in the aspiration pipette during aspiration process. Fig 1 (f) shows a photomicrograph of the ICM after removing from the blastocyst. Fig 1 (g) shows a photomicrograph of the remaining trophectoderm and zona pellucida remaining after ICM isolation. Fig 2 (a) shows a photomicrograph of another human blastocyst, secured with glass holding pipette such that the ICM is at 3 O' Clock position. Fig 2 (b) shows a photomicrograph of slight protrusion of inner cell mass after zona and trophectoderm is laser ablated. Fig 2 (c) shows a photomicrograph of the aspiration pipette being position close to the ICM after ablating the zona and neighboring trophectoderm cells with laser. Fig 2 (d) shows a photomicrograph of ICM being aspirated with the aspiration pipette by gentle suction. Fig 2 (e) shows a photomicrograph of large portion of ICM in the aspiration pipette. Fig 2 (f) shows a photomicrograph of the ICM after removing from the blastocyst. Fig 2 (g) shows a photomicrograph of the trophectoderm and zona pellucida left after the isolation of ICM from the blastocyst. Fig 3 (a) shows a photomicrograph of isolated inner cell mass in culture seeded on primary mouse embryonic fibroblast feeder layer (day 3). Fig 3 (b) shows a photomicrograph of isolated, inner cell mass in culture on primary mouse embryonic fibroblast feeder layer (day 7) Fig 4 shows a photomicrograph of isolated ICM in culture on the primary mouse embryonic fibroblast feeder layer (day 5) another embryo. Fig 5 shows a photomicrograph of embryonic stem cell line derived from inner cell mass isolated by laser ablation method.. One skilled in the art will appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned therein above. The instant invention has been shown and described herein in what is considered to be the most practical and preferred embodiment. It is recognized, that, departures may be made therefrom within the scope of the invention. It is to understood that the invention is not limited to the particulars disclosed and extends to all equivalents within the scope of the scope of the claims. We Claim 1. A method for establishing cell lines from the inner cell mass of a human blastocyst, comprising the steps of: (a) isolating a blastocyst; comprises zona pellucida, trophectoderm and inner cell mass. (b) creating an aperture in the blastocyst by laser ablation; and (c) Isolating cells of the inner cell mass from the blastocyst through the aperture. 2. The method of claim 1, wherein the aperture is through the zona pellucida. 3. The method of claim 1, wherein the aperture is through the zona pellucida and the trophectoderm. 4. The method of claim 1, wherein the laser ablation is achieved using a non-contact diode laser. 5 The method of claim 4, wherein the non-contact diode laser is a continuous 1.48 urn diode laser. 6. The method of claim 1, wherein cells of the inner cell mass are isolated by aspiration using an aspiration pipette introduced through the aperture. 7. The method of claim 1, wherein isolating cell from the inner cell mass is carried out in the absence of animal generated antibodies and sera. 8. The method of claim 1, further comprising the steps of: (a) culturing the cells of the inner cell mass in the presence of an embryonic stem cell medium and an feeder layer to produce inner cell mass derived masses; and (b) culturing the inner cell mass derived masses to obtain an isolated human embryonic stem cell line. 9. The method of claim 8, wherein the embryonic stem cell medium comprises: (a) Dulbecco's modified Eagle's medium with high glucose content in an amount from about 70% to about 90%, and without sodium pyruvate. (b) fetal bovine serum in an amount from 10% to 30% of the volume of the embryonic stem cell medium; (c) beta-mercaptoethanol (d) non-essential amino acids (e) L-glutamine and (f) basic fibroblast growth factor 10. The method of claim 8, further comprising dissociating the inner cell mass derived masses of step (a) mechanically or using any other suitable alternate method and re-plating the dissociated cells of the inner cell mass derived masses on feeder layer. 11. The method of claim 1, wherein the isolated blastocyst of step (a) is placed in conventional Embryo biopsy medium. The method of claim 11, wherein the Embryo biopsy medium is with or without Ca++/Mg++. The method of claim 1, further comprising a micromanipulator system comprising a microscope with a heated stage, a holding pipette, an aspiration pipette, and air syringes, wherein the isolated blastocyst of step (a) is placed on the heated stage, the micromanipulator system is abjusted so that the is at the center of the microscope field, and the blastocyst is secured with the holding pipette by suction through the air syringe, so that the inner cell mass is opposite the holding pipette. The method of claim 2, wherein the aperture is generated by laser ablation using a 1.48 Mm diode laser. The method of claim 3, wherein the aperture is generated by laser ablation using a 1.48 urn diode laser, and is adjacent to the inner cell mass. The method of claim 6, wherein the cells of the inner cell mass are washed one or more times in embryonic stem cell media and plated on a feeder layer in the presence of embryonic stem cell media. The method of claim 16, where in the feeder layer is of murine or human origin. The method of claim 17, wherein the feeder layer is an embryonic fibroblast feeder layer. 19. The method of claim 1, wherein the isolated cells of the inner cell mass are cultured under feeder free conditions. 20. The method of claim 19, wherein the feeder free conditions comprise an extracellular matrix. 21. The method of claim 20, wherein the feeder free conditions further comprise conditioned medium. 22. The method of claim 1, wherein the isolated blastocyst is the product of in vitro fertilization. 23. The method of claim 1, wherein the isolated blastocyst is the product of intracytoplasmic sperm injection. 24. The method of claim 8, where in the feeder layer is of murine or human origin. 25. The method of claim 8, wherein the feeder layer is an embryonic fibroblast feeder layer. 26. The method of claim 10, where in the feeder layer is of murine or human origin. 27. The method of claim 10, wherein the feeder layer is an embryonic fibroblast feeder layer. 28. The method of. claim 1, further comprising culturing the cells of the inner cell mass obtained from blastocyst to establish human embryonic stem cell line. 29. The method of claim 28, wherein the blastocyst comprises zona pellucida, trophectoderm and inner cell mass. 30. The method of claim 29, wherein the inner cell mass is isolated by aspiration through the aperture created through the zona pellucida and trophectoderm by laser ablation. 31. The method of claim 30, wherein the laser ablation is achieved using a non-contact diode laser. 32. The method of claim 28, wherein the human embryonic stem cell line is established in the absence of animal generated antibodies and sera. 33. The method of claim 28, further comprises plating the cells of the inner cell mass on a feeder layer or in feeder free condition, wherein inner cell mass-derived cell masses are formed. 34. The method of claim 33, wherein the feeder layer is an embryonic fibroblast feeder layer. 35. The method of claim 33, where in the feeder layer is of murine or human origin. 36. The method of claim 33, wherein the inner cell mass-derived cell masses are dissociated and replated on a feeder layer. 37 The method of claim 28, optionally further comprises plating the cells of the inner cell mass in a feeder free condition, wherein inner cell mass-derived cell masses are ' formed. 38. The method of claim 37, wherein the feeder free condition comprises plating the cells of the inner cell mass on an extracellular matrix. 39. The method of claim 38, wherein the cells are cultured in the presence of conditioned medium. 40 The method of claims 37, wherein the inner cell mass-derived cell masses are dissociated and replated on an extracellular matrix. 41. The method of claim 40, wherein the cells are cultured in the presence of conditioned medium. 42. The method of claim 1, optionally further comprising the steps of: (a) culturing the cells of the inner cell mass under feeder free conditions to produce inner cell mass derived masses; and (b) culturing the inner cell mass derived masses to produce an isolated human embryonic stem cell line. 43. The method of claim 42, wherein the feeder free conditions comprise an extracellular matrix. The method of claim.43, wherein the feeder free conditions further comprise conditioned medium. The method of claim 42, further comprising mechanically dissociating the inner cell mass derived masses of step (a) and re-plating the mechanically dissociated cells of the inner cell mass derived masses under feeder free conditions. The method of claim 45, wherein the feeder free conditions comprise an extracellular matrix. The method of claim 46, wherein the feeder free conditions further comprise conditioned medium. Dated this 29th day of January, 2003

Full Text

FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE SPECIFICATION
SECTION 10
" ISOLATION OF INNER CELL MASS FOR THE ESTABLISHMENT OF HUMAN EMBRYONIC STEM CELL (hESC) LINES "
RELIANCE LIFE SCIENCES PVT.LTD. an Indian Company having its Registered office at Chitrakoot, 2nd Floor, Shree Ram Mills Compound, Ganpath Rao Kadam Marg, Worli, Mumbai -400 013, Maharashtra, India.
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-

GRANTED

ORIGINAL

107/MUM/2003

Title
Isolation of inner cell mass for the establishment of human embryonic stem cell (hESC) lines.
Related Application:
This application claims priority to the PCT application No PCT/IN02/00168 dated August 20, 2002.
Field of the Invention:
The present invention relates to a method of isolation of inner cell mass (ICM) derived from embryo for establishing human embryonic stem cell (hESC) lines, using a non-contact diode laser technique.
Background of the invention
The isolation of human stem cells offers the promise of a remarkable array of novel therapeutics. Biologic therapies derived from such cells through tissue regeneration and repairs as well as through targeted delivery of genetic material are expected to be effective in the treatment of a wide range of medical conditions. Efforts to analyze and assess the safety of using human stem cells in the clinical setting are vitally important to this endeavor.
Embryonic stem (ES) cells are the special kind of cells that can both duplicate themselves (self renew) and produce differentiated functionally specialized cell types. These stem cells are capable of becoming almost all of the specialized cells of the body

and thus, may have the potential to generate replacement cells for a broad array of tissues and organs such as heart, pancreas, nervous tissue, muscle, cartilage and the like.
Stem cells have the capacity to divide and proliferate indefinitely in culture. Scientists use these two properties of stem cells to produce seemingly limitless supplies of most human cell types from stem cells, paving the way for the treatment of diseases by cell replacement. In fact, cell therapy has the potential to treat any disease that is associated with cell dysfunction or damage including stroke, diabetes, heart attack, spinal cord injury, cancer and AIDS. The potential of manipulation of stem cells to repair or replace diseased or damaged tissue has generated a great deal of excitement in the scientific, medical and/ biotechnology investment communities.
ES cells from various mammalian embryos have been successfully grown in the laboratory. Evans and Kaufman (1981) and Martin (1981) showed that it is possible to derive permanent lines of embryonic cells directly from mouse blastocysts. Thomson et al., (1995 and 1996) successfully derived permanent cell lines from rhesus and marmoset monkeys. Pluripotent cell lines have also been derived from pre-implantation embryos of several domestic and laboratory animal species such as bovines (Evans et al., 1990) Porcine (Evans et al., 1990, Notarianni et al., 1990), Sheep and goat (Meinecke-Tillmann and Meinecke, 1996, Notarianni et al., 1991), rabbit (Giles et al.„, 1993, Graves et al., 1993) Mink (Sukoyan et al., 1992) rat (lannaccona et al., 1994) and Hamster (Doetschman et al., 1988). Recently, Thomson et al (1998) and Reubinoff et al

(2000) have reported the derivation of human ES cell lines. These human ES cells resemble the rhesus monkey ES cell lines.
ES cells are found in the ICM of the human blastocyst, an early stage of the developing embryo lasting from the 4th to 7th day after fertilization. The blastocyst is the stage of embryonic development prior to implantation that contains two types of cells viz.
1. Trophectoderm: outer layer which gives extra embryonic membranes.
2. Inner cell mass (ICM): which forms the embryo proper.
In normal embryonic development, ES cells disappear after the 7th day and begin to form the three embryonic tissue layers. ES cells extracted from the ICM during the blastocyst stage, however, can be cultured in the laboratory and under the right conditions proliferate indefinitely. ES cells growing in this undifferentiated state retain the potential to differentiate into cells of all three embryonic tissue layers. Ultimately, the cells of the inner cell mass give rise to all the embryonic tissues. It is at this stage of embryogenesis, near the end of first week of development, that ES cells can be derived from the ICM of the blastocyst.
The ability to isolate ES cells from blastocysts and grow them in culture seems to depend in large part on the integrity and condition of the blastocyst from which the cells are derived. In short, the blastocyst that is large and has distinct inner cell mass tend to yield ES cells most efficiently. Several methods have been used for isolation of inner

cell mass (ICM) for the establishment of embryonic stem cell lines. Most common methods are as follows:
Natural hatching of the blastocyst:
In this procedure blastocyst is allowed to hatch naturally after plating on the feeder layer. The hatching of the blastocyst usually takes place on day 6. The inner cell mass (ICM) of the hatched blastocyst develops an outgrowth. This outgrowth is removed mechanically and is subsequently grown for establishing embryonic stem cell lines. However, this procedure has few disadvantages. Firstly, Trophectoderm cells proliferate very fast in the given culture conditions and many a times, suppress the outgrowth of inner cell mass. Secondly, while removing the outgrowth of the inner cell mass mechanically, there is a chance of isolating trophectoderm cells. Thirdly, the percentage of blastocysts hatching spontaneously in humans is very low.
Microsurgery:
Another method of isolation of inner cell mass is mechanical aspiration called microsurgery. In this process, the blastocyst is held by the holding pipette using micromanipulator system and positioned in such a way that the inner cells mass (ICM) is at 9 O'Clock position. The inner cell mass (ICM) is aspirated using a biopsy needle which is beveled shape and is inserted into the blastocoel cavity. This procedure too is disadvantageous as the possibility to isolate the complete inner cell mass is low and many a time cells get disintegrated. It is a very tedious procedure and may cause severe damage to the embryo. The operation at the cellular level requires tools with micrometer precision, thereby minimizing damage and contamination.

Immunosurgery:
This is a commonly used procedure to isolate inner cell mass (ICM). The inner cell mass (ICM) is isolated by complement mediated lysis. In this procedure, the blastocyst is exposed either to acid tyrode solution or pronase enzyme solution in order to remove the zona pellucida (shell) of blastocyst. The zona free embryo is then exposed to human surface antibody for about 30 min to one hour. This is followed by exposure of embryos to guinea pig complement in order to lyse the frophectoderm. This complement mediated lysed trophectoderm cells are removed from inner cell mass (ICM) by repeated mechanical pipetting with a finely drawn Pasteur pipette. All the embryonic stem cell lines reported currently in the literature have been derived by this method. However, this method has several disadvantages. Firstly, the embryo is exposed for a long time to acid tyrode or pronase causing deleterious effects on embryo, thereby reducing the viability of embryos proper. Secondly, it is time consuming procedure as it takes about 1.5 to 2.0 hours. ( Narula et al.,1996). Thirdly, the yield of inner cell mass (ICM) per blastocyst is low. Fourthly, critical storage conditions are required for antibody and complement used in the process. Lastly, it involves the risk of transmission of virus and bacteria of animal origin to humans, as animal derived antibodies and complement are used in the process. In this process, two animal sera are used. One is rabbit antihuman antiserum and the other is guinea pig complement sera.
The human cell lines studied to date are mainly derived by using a method of immunosurgery, where animal based antisera and complement was used.
Other possible disadvantages of the existing cell lines are as follows:-

1 Use of feeder cells for culturing the human embryonic stem cell (hESC) lines
produces mixed cell population that require the Embryonic stem cells (ESC) to be separated from feeder cell components and this impairs scale up.
2. Embryonic stem cells (ESC) get contaminated by transcripts from feeder cells and cannot be used on a commercial scale. It can be used only for research purposes.
Geron established a procedure where human Embryonic Stem Cell (hESC) line was cultured in the absence of feeder cells (Xu et.al., 2001). The hESC were cultured on an extracellular matrix in a conditioned medium and expanded in this growth environment in an undifferentiated state. The hESC contained no xenogenic components of cancerous origin from other cells in the culture. Also, the production of hESC cells and their derivatives were more suited for commercial production. In this process, there was no need to produce feeder cells on an ongoing basis to support the culture, and the passaging of cells could be done mechanically. However, the main disadvantage of this procedure is that the inner cell mass (ICM) is isolated by immunosurgery method, wherein the initial derivation of Embryonic Stem Cells is carried out using feeder layer containing xenogenic components. This raises the issue of possible contamination with animal origin viruses and bacteria.
In order to simplify the procedure of inner cell mass isolation and to make it safe, the scientists of the present invention have come out with a novel method of isolation of the

inner cell mass using a non-contact laser, wherein, the use of animal based antisera and complement have been eliminated.
Use of Laser technique in Assisted Reproduction:
With the advent of assisted reproductive technologies (ART), several methods have been used for improving fertilization, facilitating blastocyst hatching (Cohen et al, 1990) and performing blastomere biopsy (Tarin and Handyside, 1993). Commonly used methods are chemical (Gordon and Talansky, 1986), mechanical (Depypere et al., 1988) and laser (Feichtinger et al., 1992) so as to produce holes in the zona pellucida (Gordon, 1988). Recently, an infrared 1.48 pm diode laser beam focused through a microscope objective was shown to allow rapid, easy and non-touch microdrilling of mouse and human zona pellucida and high degree of accuracy was maintained under conventional culture conditions (Rink et al., 1994). The drilling effect was shown due to a highly localized heat-dependent disruption of the zona pellucida glycoprotein matrix (Rink et al., 1996). Contrary to the detrimental effect on compacted mouse embryos induced by the 308 nm xenon-chlorine excimer laser (Neev et al., 1993), the drilling process in the infrared region did not affect embryo survival in mice (Germond et al., 1995) or in humans (Antinori et al., 1994).
Currently, laser is being investigated as a tool to aid fertilization and in assisted hatching. Recent reports show that use of 1.48 pm diode laser for microdrilling mouse zona pellucida is highly safe and does not affect neuro-anatomical and neurochemical properties in mice and also improves fertilization (Germond et al., 1996). Obruca and colleagues first reported the success of laser-assisted hatching in human IVF in 1994. In

this study, a 20- to 30-micron hole was made in the zona pellucida ( ZP ) when the embryos were at the two- to four-cell stage, and embryos were transferred immediately. Patients with previous IVF failures from two separate centers were included in this study. There was a higher implantation rate per embryo in the laser-assisted hatching group (14.4%) versus the control group (6%). Pregnancy rates per transfer were also improved (40% versus 16.2%).
In a separate study, Er:YAG laser was used to thin the ZP of embryos derived from patients undergoing repeated IVF. Using a laser for thinning the ZP, embryologists are able to achieve accurate reduction of the ZP by 50%, which is very difficult with acidic Tyrode's solution. Presence of Acid Tyrode's solution near the embryo may also be detrimental. The rate of clinical pregnancies in the laser-hatched group was 42.7%, as compared to 23.1% in the control unhatched group. Since this data looked promising, the indication of laser-assisted hatching was extended. Women undergoing IVF for the first time yielded 39.6% clinical pregnancy rate in the laser-treated group versus a 19% rate in the control unhatched group (Parikh et al 1996).
During the last decade there has been ongoing research on the isolation of inner cell mass (ICM), as it is useful in establishing embryonic stem cell lines which in turn have the ability to develop into most of the specialized cells in the human body including blood, skin, muscle and nerve cells. They also have the capacity to divide and proliferate indefinitely in culture.

The present invention involves the isolation of inner cell mass (ICM), using laser ablation technique without undergoing the cumbersome procedure of immunosurgery. Hence, in the present invention, the use of animal derived antibodies or sera are eliminated and the procedure is safe, simple, rapid and commercially viable.
The present invention, obviates the shortcomings associated with the conventional methods of isolation of inner cell mass (ICM). The inner cell mass (ICM) isolated by the present invention is found to be intact without causing any destruction or damage to the cells. The present invention thus provides a quick reliable and non-invasive method for isolation of inner cell mass (ICM). It also completely ruptures the trophectoderm thereby minimizing the contamination of inner cell mass (ICM), thus ensuring the purity of inner cell mass (ICM).
REFERENCES
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3. Depypere HT, McLaughlin KJ, Seamark RF et al (1988). Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse. J. Reprod Fertil. 84:205-211.
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10. Germond M, Nocera D, Senn A. Rink K. et al (1995). Microdissection of mouse and human zona pellucida using 1.48 microns diode laser beam: efficacy and safety of the procedure. Fertil Steril. 64: 604-611.
11. Germond M, Nocera D, Senn A, Rink A et al (1996). Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 micron diode laser beam. Hum Reprod. 11: 1043-1048
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Objects of the Invention
1. It is an object of the present invention, to develop a process of isolation of inner cell mass, using laser ablation technique, without undergoing the cumbersome procedure of immunosurgery.
2. It is another object of the present invention to isolate ICM using laser ablation technique without using any animal generated antibodies and sera, thereby preventing the possibility of transmission of animal organism to human and thus can safely be used on commercial scale.
3. It is another object of the present invention to isolate inner cell mass (ICM) from blastocyst stage of a mammalian embryo using a non-contact diode laser.
4. It is another object of the present invention to isolate inner cell mass (ICM) by simple, shorter and easily feasible way without affecting/destroying the inner cell mass (ICM).
5. It is still another object of the present invention to ensure the purity of inner cell mass (ICM) by ablating completely trophectoderm thereby minimising the contamination of inner cell mass (ICM).

6. It is still another object of the present invention to isolate inner cell mass (ICM) of high yield and purity as compared to the inner cell mass (ICM) isolated by the conventional methods.
These and other objects of the invention will become more readily apparent from the ensuing description.
Details of Invention:
The present invention relates to isolation of inner cell mass, using laser ablation of zona pellucida (ZP) and trophectoderm (TE) and aspiration of inner cell mass for establishing embryonic stem cell lines. In the present invention, the non contact diode laser used is highly accurate and reliable tool for cellular microsurgery. The system incorporates the latest in fiber optic technology to provide the most compact laser system currently available. The 1.48 pm diode non-contact Laser System is mounted/implanted via the epifluorescence port to inverted microscope fitted with micromanipulators. A pilot laser is used to target the main ablation laser and a series of LEDs inform the user when the laser is primed and is ready to fire. A two-second-operation window is used to reduce the possibility of accidentally firing the laser. The spot diameter of the laser can be varied according to the hole size required.
Couples undergoing in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) treatment voluntarily donate surplus human embryos. These embryos are used for research purposes after taking the informed written, voluntary consent from these couples. In the present invention, blastocyst stage embryos are taken for the isolation of

inner cell mass. The blastocyst is placed in a petridish in a microdroplets of embryo biopsy medium with or without Ca++/Mg++ and is covered with mineral oil. The micromanipulator is set up to perform the embryo biopsy procedure. The blastocyst is placed in embryo biopsy medium and the petridish containing the blastocyst is placed on the heated stage of the microscope. The blastocyst is positioned at the center of the field. The blastocyst is immobilized on to the holding pipette in such a way that the inner cell mass is at 3 O' Clock position. The zona pellucida and trophectoderm close to inner cell mass is positioned on the aiming spot of the laser beam. A small portion of zona pellucida and trophectoderm is laser ablated. Biopsy pipette is then gently inserted through the hole in the zona pellucida and trophectoderm and the inner cell mass is gently aspirated. After isolation of the complete inner cell mass, the cells are given several washes with embryonic stem cell (ESC) medium. The cells are then plated on to coated plate in the presence of embryonic stem cell medium, with or without feeder layer for establishing embryonic stem cell lines. The embryonic stem cells were then characterized for cell surface markers such as SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4 and alkaline phosphatase. The embryonic stem cell lines are also karyotyped.
a) Development of blastocyst in vitro:
Institutional Ethics Committee approval has been obtained before initiation of this study. Prior written consent was taken from individual donor for the donation of surplus embryos for this study after completion of infertility treatment.
Protocol generally used for infertility patients for obtaining viable embryo is as follows:

The ovarian superovulation began with gnRH agonist analog suppression daily starting in the mid-luteal phase and administered in doses of 500-900 mgs for about 9-12 days. Ovarian stimulation was started after adequate ovarian suppression with human menopausal gonadotropins (hMG) or recombinant follicle stimulating hormone (FSH) (Gonal-F, Recagon) in appropriate doses depending on the age and ovarian volume. The dose was also adjusted as necessary to produce controlled ovarian stimulation. Serum beta-estradiol (E2) measurements were carried out as required. Transvaginal ultrasound was performed daily from cycle day 7 onward. Human Chorionic gonadotropin 5000-10000 I.U. was administered when three or more follicles were at least 17 mm in largest diameter. Transvaginal aspiration was performed 34-36 h later. Oocytes were then subjected to intracytoplasmic sperm injection.
A holding pipette was used to secure the egg. Motile sperm were placed in a drop of polyvinyl pyrolidone (PVP) solution and overlaid with mineral oil. An injection needle was used to pierce the zona pellucida at about 3 O' Clock position. The selected spermatozoon was immobilized by cutting the tail with the injection micropipette. The oocyte was secured with holding pipette and spermatozoon was injected directly into the center of the oocyte.
Oocytes were checked after 16-18 hours of culture for fertilization. At this point the fertilized oocyte had pro-nuclei (also called one cell embryo). One-cell embryos were then transferred into pre-equilibrated fresh ISM-1 medium or any other suitable embryo culture medium and incubated at 37 ° C in a 5% C02 in air. The next day embryos were transferred into ISM-2 medium or any oVneT suitabie embryo culture medium. Every

alternate day embryos were transferred into fresh ISM-2 medium or any other suitable embryo culture medium. From day 5 onward embryos were checked for the blastocyst development. After the treatment is over, the surplus blastocysts were donated by the couples for this research work.
b) Setting up of the Laser:
The present invention relates to describing a unique method for inner cell mass isolation for establishment of embryonic stem cells using the non-contact diode laser. The laser is highly accurate and reliable tool for cellular microsurgery. The system incorporates the latest in fiber optic technology to provide the most compact laser system currently available. The 1.48 pm non-contact diode Laser System was mounted via the epifluorescence port to inverted microscope fitted with micromanipulators.
A pilot laser was used to target the main ablation laser and a series of LED's inform the user when the laser is primed and ready to discharge the laser beam. A two-second-operation window was used to reduce the possibility of accidentally firing the laser. The spot diameter of the laser can be varied according to the ablation size required.
c) Laser Ablation and isolation of inner cell mass.
The blastocyst was individually placed in a microdroplet of biopsy medium (with or without Ca *7Mg ++ in a petri dish. The embryo was immobilized with the holding pipette in such a way that the inner cell mass remained at 3 O' Clock position and the zona pellucida and trophectoderm close to inner cell mass positioned on the aiming spot. A 1.48 urn diode laser was used to create an aperture in the Zona Pellucida (ZP), which is

a glycoprotein layer protecting the oocyte/embryo. At this wavelength, the hole was induced by a local thermo-dissolution of the glycoprotein matrix. Once the zona pellucida was dissolved, trophectoderm cells were ablated by giving 3 or more pulses to cause photolysis. After ablation of both zona pellucida and trophectoderm, the aspiration pipette was introduced through laser-ablated hole and ICM was removed by gentle aspiration, with the aspiration pipette of required diameter.
d) Culturing of human Embryonic Stem Cells (hESC)
Prior to culturing, the aspirated ICM was washed thoroughly in ES medium. Given below is the procedure when the invention was carried out using feeder layer. In this process, the inner cell mass was cultured in tissue culture plate in the presence of inactivated mouse embryonic fibroblast feeder layer. Mouse embryonic fibroblast feeder layer was preferably obtained from Theiler stage mouse embryos i.e. 21 &22 (12.5 -15 days port coital) C57BL/6 mice or C57BL/6XSJL F-1 mice or out bred CD1 mice or from human amniotic fluid and used as a feeder layer. The fibroblast feeder layer was inactivated by gamma irradiation (3500 rads) or treatment with colchicine. The inactivated mouse embryonic fibroblast feeder layer was cultured on 0.5% gelatin coated plate with ES medium consisting of Dulbecco's modified Eagle's medium without Sodium pyruvate with high glucose contain (70-90%), Fetal bovine serum (10-30%), beta-mercaptoethanol, non-essential amino acids, L- Glutamine, basic fibroblast growth factor. After 4-7 days, ICM derived masses were removed from outgrowth with sterile fire polished pipette and were dissociated mechanically or with any other alternative method and plated on fresh feeder cells.

Established cell lines were karyotyped and characterized for several surface markers such as SSEA-1, SSEA-3, SSEA-4, OCT-4, Alkaline phosphatase, TRA-1-81, TRA-1-60 as described by Thomson et al., (1998), Reubinoff et al., (2000).
Examples
The following examples are intended to illustrate the invention but do not limit the scope thereof.
Example I:
Total of 24 blastocyst stage human embryos were used for the isolation of inner cell mass. Embryos were washed several times in blastocyst culture medium (ISM-2 medium, Medicult, Denmark). Biopsy dish was prepared containing 50 |il microdroplets of Ca++/ Mg++ free embryo biopsy medium (EB 10 medium, Scadinavian) overlaid with mineral oil and individual blastocyst was then placed in the microdroplet. Micromanipulator was set up. A holding pipette with outer diameter 75 jam and inner diameter 15 pm was used to secure the embryo. Biopsy pipette with an outer diameter of roughly 49 pm and inner diameter 35 prn was used for aspiration of inner cell mass. A pilot laser was used to target the main ablation laser. Blastocyst was immobilized with the holding pipette in such a way that inner cell mass remained at 3 O' clock position and the zona pellucida and trophectoderm close to inner cell mass positioned to the aiming spot. The hole was induced by a local thermo-dissolution of the zona. Trophectoderm cells were ablated by giving 3 pulses to cause photolysis. After ablation of both the zona pellucida and trophectoderm, the biopsy pipette was introduced through laser ablated hole and inner cell mass was removed. Inner cell mass was then washed

several times in ES medium and placed in tissue culture plate in the presence or absence of feeder layer.
The efficiency of the present invention i.e. isolation of inner cell mass by laser ablation method was compared with isolation of inner cell mass with conventional method i.e. immunosurgery.
The comparative data is given below;
Table 1: Summary of hESC lines developed using Laser ablation Technique of the
present invention with the use of mouse feeder cells.
No. of Total inner With mouse feeder cells
blastocyst cell mass
used for laser removed No- of No of ES cell lines established
ablation ICM used
24 18 14 4
Example 2:
Similarly, experiments were conducted with conventional method of isolation of inner cell mass i.e. using immunosurgery as given below:
A total of 21 human blastocyst were used for isolation of inner cell mass. Embryos were washed several times with blastocyst culture medium (ISM-2 medium) and followed by ES medium. Individual blastocyst was then placed in 50 pl micodrops of 1:50 anti-human antibody (Sigma) for 30 minutes at 37 o C and 5% C02 in air. After incubation blastocyst

were then washed four times with ES medium! Blastocysts were then placed in 50 (il of microdroplets of guinea pig complement at the concentration of 1:10 for 10 minutes at 37°C and 5% C02 in air. After incubation blastocyst were washed several times in ES medium using fine bore glass pipette in order to remove trophectoderm. Isolated inner . cell mass was then washed with ES medium and cultured in tissue culture plate in the presence or absence of feeder cells. Data are presented in the table:
Table 2: Summary of hESC lines developed using immunosurgery with / without the use of mouse feeder cells.
No. of Total With mouse feeder cells Without mouse feeder cells
blastocyst Inner cell
used for mass No. of No of ES cell No. of ICM used No. of ES
laser removed ICM used lines cell lines
ablation established established
21 14 .12 3 2 0
Although the isolation of inner cell mass using both the methods did not show any significant difference, the isolation of inner cell mass by laser ablation has distinct advantage. This method eliminates the use of antibodies and sera of animal origin. Isolation of inner cell mass by laser ablation method of the present invention can be cultured in the presence or absence of feeder layer. However, culturing of inner cell mass in a feeder free condition will further eliminate the possibilities of contamination of ES cell lines with animal viruses or bacteria and can be commercially utilized for human

transplantation studies. In the current experiments, efforts were also made to establish ES cell line in the absence of feeder cells.
Detailed description of the preferred embodiments:
A preferred embodiment of the invention is illustrated in the accompanying photomicrographs.
Fig 1 (a) to 1 (g) of the present invention, pertains to the isolation of inner cell mass (ICM) from the blastocyst of one embryo and Fig 2(a) to 2(g) pertains to the isolation of inner cell mass (ICM) from the blastocyst of another embryo. Fig 3,4,and 5 pertains to culturing of ICM on feeder cells at different stages.
Fig 1. (a) shows a photomicrograph of human blastocyst, secured with glass holding pipette such that the ICM is at 3 O' Clock position.
Fig 1 (b) shows a photomicrograph wherein part of zona pellucida and trophectoderm ablated with laser (arrow).
Fig 1 (c) shows a photomicrograph of aspiration pipette close to the blastocyst following zona and trophectoderm ablation.
Fig 1 (d) shows a photomicrograph of beginning of aspiration of ICM with aspiration pipette.

Fig 1 (e) shows a photomicrograph of large portion of ICM in the aspiration pipette during aspiration process.
Fig 1 (f) shows a photomicrograph of the ICM after removing from the blastocyst.
Fig 1 (g) shows a photomicrograph of the remaining trophectoderm and zona pellucida remaining after ICM isolation.
Fig 2 (a) shows a photomicrograph of another human blastocyst, secured with glass holding pipette such that the ICM is at 3 O' Clock position.
Fig 2 (b) shows a photomicrograph of slight protrusion of inner cell mass after zona and trophectoderm is laser ablated.
Fig 2 (c) shows a photomicrograph of the aspiration pipette being position close to the ICM after ablating the zona and neighboring trophectoderm cells with laser.
Fig 2 (d) shows a photomicrograph of ICM being aspirated with the aspiration pipette by gentle suction.
Fig 2 (e) shows a photomicrograph of large portion of ICM in the aspiration pipette.
Fig 2 (f) shows a photomicrograph of the ICM after removing from the blastocyst.

Fig 2 (g) shows a photomicrograph of the trophectoderm and zona pellucida left after the isolation of ICM from the blastocyst.
Fig 3 (a) shows a photomicrograph of isolated inner cell mass in culture seeded on primary mouse embryonic fibroblast feeder layer (day 3).
Fig 3 (b) shows a photomicrograph of isolated, inner cell mass in culture on primary mouse embryonic fibroblast feeder layer (day 7)
Fig 4 shows a photomicrograph of isolated ICM in culture on the primary mouse embryonic fibroblast feeder layer (day 5) another embryo.
Fig 5 shows a photomicrograph of embryonic stem cell line derived from inner cell mass isolated by laser ablation method..
One skilled in the art will appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned therein above. The instant invention has been shown and described herein in what is considered to be the most practical and preferred embodiment. It is recognized, that, departures may be made therefrom within the scope of the invention. It is to understood that the invention is not limited to the particulars disclosed and extends to all equivalents within the scope of the scope of the claims.

We Claim
1. A method for establishing cell lines from the inner cell mass of a human blastocyst, comprising the steps of:
(a) isolating a blastocyst; comprises zona pellucida, trophectoderm and inner cell mass.
(b) creating an aperture in the blastocyst by laser ablation; and
(c) Isolating cells of the inner cell mass from the blastocyst through the aperture.

2. The method of claim 1, wherein the aperture is through the zona pellucida.
3. The method of claim 1, wherein the aperture is through the zona pellucida and the trophectoderm.
4. The method of claim 1, wherein the laser ablation is achieved using a non-contact diode laser.
5 The method of claim 4, wherein the non-contact diode laser is a continuous 1.48 urn
diode laser.
6. The method of claim 1, wherein cells of the inner cell mass are isolated by aspiration using an aspiration pipette introduced through the aperture.

7. The method of claim 1, wherein isolating cell from the inner cell mass is carried out in the absence of animal generated antibodies and sera.
8. The method of claim 1, further comprising the steps of:

(a) culturing the cells of the inner cell mass in the presence of an embryonic stem cell medium and an feeder layer to produce inner cell mass derived masses; and
(b) culturing the inner cell mass derived masses to obtain an isolated human embryonic stem cell line.
9. The method of claim 8, wherein the embryonic stem cell medium comprises:
(a) Dulbecco's modified Eagle's medium with high glucose content in an amount from about 70% to about 90%, and without sodium pyruvate.
(b) fetal bovine serum in an amount from 10% to 30% of the volume of the embryonic stem cell medium;
(c) beta-mercaptoethanol
(d) non-essential amino acids
(e) L-glutamine and
(f) basic fibroblast growth factor

10. The method of claim 8, further comprising dissociating the inner cell mass derived masses of step (a) mechanically or using any other suitable alternate method and re-plating the dissociated cells of the inner cell mass derived masses on feeder layer.
11. The method of claim 1, wherein the isolated blastocyst of step (a) is placed in conventional Embryo biopsy medium.

The method of claim 11, wherein the Embryo biopsy medium is with or without Ca++/Mg++.
The method of claim 1, further comprising a micromanipulator system comprising a microscope with a heated stage, a holding pipette, an aspiration pipette, and air syringes, wherein the isolated blastocyst of step (a) is placed on the heated stage, the micromanipulator system is abjusted so that the is at the center of the microscope field, and the blastocyst is secured with the holding pipette by suction through the air syringe, so that the inner cell mass is opposite the holding pipette.
The method of claim 2, wherein the aperture is generated by laser ablation using a 1.48 Mm diode laser.
The method of claim 3, wherein the aperture is generated by laser ablation using a 1.48 urn diode laser, and is adjacent to the inner cell mass.
The method of claim 6, wherein the cells of the inner cell mass are washed one or more times in embryonic stem cell media and plated on a feeder layer in the presence of embryonic stem cell media.
The method of claim 16, where in the feeder layer is of murine or human origin.
The method of claim 17, wherein the feeder layer is an embryonic fibroblast feeder layer.

19. The method of claim 1, wherein the isolated cells of the inner cell mass are cultured under feeder free conditions.
20. The method of claim 19, wherein the feeder free conditions comprise an extracellular matrix.
21. The method of claim 20, wherein the feeder free conditions further comprise conditioned medium.
22. The method of claim 1, wherein the isolated blastocyst is the product of in vitro fertilization.
23. The method of claim 1, wherein the isolated blastocyst is the product of intracytoplasmic sperm injection.
24. The method of claim 8, where in the feeder layer is of murine or human origin.
25. The method of claim 8, wherein the feeder layer is an embryonic fibroblast feeder layer.
26. The method of claim 10, where in the feeder layer is of murine or human origin.
27. The method of claim 10, wherein the feeder layer is an embryonic fibroblast feeder layer.
28. The method of. claim 1, further comprising culturing the cells of the inner cell mass obtained from blastocyst to establish human embryonic stem cell line.

29. The method of claim 28, wherein the blastocyst comprises zona pellucida, trophectoderm and inner cell mass.
30. The method of claim 29, wherein the inner cell mass is isolated by aspiration through the aperture created through the zona pellucida and trophectoderm by laser ablation.
31. The method of claim 30, wherein the laser ablation is achieved using a non-contact diode laser.
32. The method of claim 28, wherein the human embryonic stem cell line is established in the absence of animal generated antibodies and sera.
33. The method of claim 28, further comprises plating the cells of the inner cell mass on a feeder layer or in feeder free condition, wherein inner cell mass-derived cell masses are formed.
34. The method of claim 33, wherein the feeder layer is an embryonic fibroblast feeder layer.
35. The method of claim 33, where in the feeder layer is of murine or human origin.
36. The method of claim 33, wherein the inner cell mass-derived cell masses are dissociated and replated on a feeder layer.
37 The method of claim 28, optionally further comprises plating the cells of the inner cell mass in a feeder free condition, wherein inner cell mass-derived cell masses are ' formed.

38. The method of claim 37, wherein the feeder free condition comprises plating the cells of the inner cell mass on an extracellular matrix.
39. The method of claim 38, wherein the cells are cultured in the presence of conditioned medium.
40 The method of claims 37, wherein the inner cell mass-derived cell masses are dissociated and replated on an extracellular matrix.
41. The method of claim 40, wherein the cells are cultured in the presence of conditioned medium.
42. The method of claim 1, optionally further comprising the steps of:
(a) culturing the cells of the inner cell mass under feeder free conditions to produce inner cell mass derived masses; and
(b) culturing the inner cell mass derived masses to produce an isolated human embryonic stem cell line.
43. The method of claim 42, wherein the feeder free conditions comprise an extracellular
matrix.

The method of claim.43, wherein the feeder free conditions further comprise conditioned medium.
The method of claim 42, further comprising mechanically dissociating the inner cell mass derived masses of step (a) and re-plating the mechanically dissociated cells of the inner cell mass derived masses under feeder free conditions.
The method of claim 45, wherein the feeder free conditions comprise an extracellular matrix.
The method of claim 46, wherein the feeder free conditions further comprise conditioned medium.
Dated this 29th day of January, 2003

Documents:

107-mum-2003-cancelled page(29-01-2003).pdf

107-mum-2003-claim(granted)-(17-12-2004).pdf

107-mum-2003-claims(granted)-(17-12-2004).doc

107-mum-2003-correspondenc ipo-(10-08-2006).pdf

107-mum-2003-correspondence(28-07-2006).pdf

107-mum-2003-drawing-(17-12-2004).pdf

107-mum-2003-form 1(29-01-2003).pdf

107-mum-2003-form 19(19-11-2003).pdf

107-mum-2003-form 2(granted)-(17-12-2004).doc

107-mum-2003-form 2(granted)-(17-12-2004).pdf

107-mum-2003-form 24(28-07-2006).pdf

107-mum-2003-form 3(08-09-2004).pdf

107-mum-2003-form 3(29-01-2003).pdf

107-mum-2003-pct ipea 409(29-01-2003).pdf

107-mum-2003-pct isa 210(29-01-2003).pdf

107-mum-2003-petiotion under rule 138(08-09-2004).pdf

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Patent Number

202293

Indian Patent Application Number

107/MUM/2003

PG Journal Number

42/2008

Publication Date

17-Oct-2008

Grant Date

10-Aug-2006

Date of Filing

29-Jan-2003

Name of Patentee

RELIANCE LIFE SCIENCES PRIVATE LIMITED

Applicant Address

CHITRAKOOT, 2ND FLOOR, GANPATRAO KADAM MARG, SHREE RAM MILLS COMPOUND, LOWER PAREL, MUMBAI,

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. FIRUZA RAJESH PARIKH	122 JOLLY MAKER III, 119,CUFF PARADE, MUMBAI- 400 025,
2	DR. SATISH MAHADEORAO TOTEY	FLAT NO.72,7TH FLOOR,A WING, NAPEROL TOWER, R. A. KIDWAI ROAD, WADALA, MUMBAI - 400 031,
3	DR. SHAILAJA ANUPAM SAXENA	811,PUNIT TOWER - II, SECTOR 11, CBD BELAPUR, NAVI MUMBAI - 700 614.

PCT International Classification Number

N/A

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA