Title of Invention

A PROCESS FOR THE PREPARATION OF THREE-DIMENSIONAL TISSUE EQUIVALENT USING MACROMASS CULTURE

Abstract The present invention provides a three-dimensional tissue equivalent for in-vivo and in-vitro uses. The there dimensional tissue equivalent of the present invention is a non-contractile cellular sheet cultured over a porous scaffold by a specially designed process wherein the cell sheet is entirely on one side of the porous sponge. In particular, the present invention provides a dermal wound dressing which comprises high cell density.
Full Text FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
PATENT OF ADDITION
(See Section 54;)
A PROCESS FOR THE PREPARATION OF
THREE-DIMENSIONAL TISSUE EQUIVALENT
USING MACROMASS CULTURE.
RELIANCE LIFE SCIENCES PVT.LTD.
an Indian Company having its Registered office at
Chitrakoot, 2nd Floor,
Ganpatrao Kadam Marg,
Shree Ram Mills Compound,
Lower Parel, Mumbai 400 013,
Maharashtra, India
The following specification describes and ascertains the nature of this invention and the manner in which it is performed: -
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is a patent of addition of Indian patent 195953, filed October 18, 2002 and claims the benefit of U.S. patent application number 20040082063 filed October 16, 2003 and PCT application WO/2005/095585 each of which are hereby incorporated by reference in its entirety for all purposes.
FIELD OF THE INVENTION
The present invention relates to the field of tissue engineering for in vivo or in vitro uses. Further more specifically, this invention relates to a non-contractile three-dimensional tissue equivalent as a dermal wound dressing and its methods of preparation. The present invention also relates to the potential therapeutic applications of the non-contractile three-dimensional tissue equivalent and its safety and efficacy evaluation in the treatment of wounds. Alternatively the three-dimensional tissue equivalent of the present invention can also be used for in vitro cytotoxicity screening of compounds.
BACKGROUND OF THE INVENTION Skin dermis
Dermal fibroblasts are cells present in the dermis of the skin, within extracellular matrix composed of collagen. The dermis provides strength and flexibility to the skin, and also is a supporting structure for blood vessels, lymphatic system, nerves, sweat glands and hair follicles. Fibroblasts are the major cell type of the dermis, which produce and maintain the extracellular matrix, which in turn supports other cell, types (Parenteau NL, Hardin-Young J, Ross RN (2000) Skin. In Principles of Tissue Engineering, 2nd Ed. Academic Press, San Diego.) The fibroblasts secrete various growth factors and cytokines and produce new extracellular matrix in the granulation tissue. Fibroblasts convert to a contractile myofibroblast phenotype, which in turn initiates contraction of the wound and epithelization takes place, leading to complete wound closure.
Etiology of wounds
Wound healing, or wound repair, is the body's natural process of regenerating dermal and epidermal tissue. When an individual is wounded, a set of events takes place in a predictable
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fashion to repair the damage. These events overlap in time and must be artificially categorized into separate steps: the inflammatory, proliferative, and maturation phases. The wound healing process of the skin is divided into the overlapping phases of inflammation, tissue formation and tissue remodeling (Clark RAF, Singer AJ (2000) Wound repair : Basic Biology to Tissue Engineering. In Principles of Tissue Engineering, 2nd Ed. Academic Press, San Diego.) Granulation tissue formation and wound contraction during normal wound healing are processes in which the dermal fibroblasts have an important role to play. In the inflammatory phase, bacteria and debris are phagocytized and removed and factors are released that cause the migration and division of cells involved in the proliferative phase.
The proliferative phase is characterized by angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction. In angiogenesis, new blood vessels grow from endothelial cells. In fibroplasia and granulation tissue formation, fibroblasts grow and form a new, provisional extracellular matrix (ECM) by secreting collagen and fibronectin. In epithelialization, epithelial cells migrate across the wound bed to cover it. In contraction, the wound is made smaller by the action of myofibroblasts, which establish a grip on the wound edges and contract themselves using a mechanism similar to that in smooth muscle cells. Unneeded cells undergo apoptosis when the cells' roles are close to complete. In the maturation and remodeling phase, collagen is remodeled and realigned along tension lines and cells that are no longer needed are removed by apoptosis.
Wounds that fail to undergo closure in a normal course of time are termed as chronic or nonhealing wounds [Lorenz HP and Longaker MT. 2003. Wounds: Biology, Pathology, and Management. Stanford University Medical Center]. Lower extremity skin ulcers can result from arterial or venous insufficiency. Clinical factors that affect the repair process in nonhealing ulcers are diabetic condition, ischemia, bacterial infection, and nutrition. In the diabetic condition, the classical risk factors for developing ulcers are peripheral neuropathy, peripheral arterial disease, and susceptibility to infection [Thuesen A. 2001. Graftskin (Apligraf®) for diabetic foot ulcers. The University of Montana SPAHS Drug Information Service 5 : 1-3.]
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Treatment of wounds
The primary goal in the treatment of diabetic foot ulcers is to obtain wound closure. Management of the foot ulcer is largely determined by its severity (grade) and vascularity, and the presence of infection. A multidisciplinary approach should be employed because of the multifaceted nature of foot ulcers and the numerous comorbidities that can occur in these patients. This approach has demonstrated significant improvements in outcomes, including reduction in the incidence of major amputation.
A mainstay of ulcer therapy is debridement of all necrotic, callus, and fibrous tissue. Unhealthy tissue must be sharply debrided back to bleeding tissue to allow full visualization of the extent of the ulcer and detect underlying abscesses or sinuses.
Topical applications have been tried for the treatment of diabetic ulcers with some success. An example is the use of placental extract, which contains various growth factors, and phenytoin for treating non-healing ulcers [Chauhan VS, Rashid MA, Pandley SS, Shukla VK. 2003. Non-healing wounds: A therapeutic dilemma. Lower Extremity Wounds 2 : 40-45.]. Another topical application, containing recombinant human platelet derived growth factor (PDGF), is Plermin, marketed by Dr. Reddy's Laboratories. PDGF is the only topically applied growth factor approved for therapeutic use [Falanga V. 2005. Advanced treatments for non-healing chronic wounds. World Wide Wounds], for which randomized controlled clinical trials have shown an acceleration in the healing of neuropathic diabetic foot ulcers by only about 15 %. Although numerous topical medications and gels are promoted for ulcer care, relatively few have proved to be more efficacious than saline wet-to-dry dressings. Topical antiseptics, such as povidone-iodine, are usually considered to be toxic to healing wounds. Topical enzymes have not been proved effective for this purpose and should only be considered as adjuncts to sharp debridement. Soaking ulcers is controversial and should be avoided because the neuropathic patient can easily be scalded by hot water.
Other growth factors have not been approved for clinical use and the results of clinical trials have not delivered the expectations generated by preclinical data [Falanga V. 2005. Advanced treatments for non-healing chronic wounds. World Wide Wounds]. A possible
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explanation for this could be that growth factors are required in combination, or a different mode of delivery is required. Cells, in this respect, are considered "smart materials" which can produce balanced mixtures of different growth factors and cytokines, and they can adapt their responses according to the environment they are in, thereby helping in repairing the affected area./damaged tissue. [Falanga V. 2005. Advanced treatments for non-healing chronic wounds. World Wide Wounds]. Hence, cell-based applications have more potential for much better results.
With the advent of the science of tissue engineering, there have been promising results shown by different skin substitutes in the efficient treatment of chronic wounds which otherwise have been difficult to heal successfully, and have often lead to requirement for amputation of the limb having the ulcer. [Eisenbud D, Huang NF, Luke S, Silberklang M. 2004. Skin substitutes and wound healing : Current status and challenges. Wounds 16:2-17. and Marston WA, Hanft J, Norwood P, Pollak R. 2003. The efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers. Diabetes Care 26 : 1701-1705.] Skin autografts are successful in effecting wound healing, but the autografting procedure is invasive, painful and could lead to a secondary non-healing wound. In the case of chronic wounds, a skin substitute that can play the role of a temporary biological dressing which would trigger tissue regeneration and wound healing by stimulating the cells in the patient's own wound bed has potential to be an effective treatment modality. The cells in the skin substitute are expected to be effective delivery systems for growth factors that would help in stimulating the healing process.
Various skin substitutes have been developed internationally for the treatment of non-healing ulcers. Examples are Apligraf® (Organogenesis Inc.), Dermagraft® (Smith & Nephew Inc.), Oasis® (Healthpoint), and EZ Derm™ (Brennen Medical Inc.). These have shown good clinical results. However there are challenges faced such as, difficult logistics of ordering and use due to requirement for cryopreservation, difficulty in maintaining cell viability, poor durability of matrix collagen when exposed to the enzyme-rich wound bed causing cells to get washed away and hence losing effect, the thickness of the matrix not allowing good enough diffusion of growth factors from the embedded cells, and these issues have lead to the
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requirement for improvement and innovative solutions. Another pertinent problem is the cost of these skin substitutes. Due to the problems associated with such skin substitutes, the inventors of the present invention have been successful in providing an improvised and an alternate solution to the skin substitutes by providing a temporary biological dressing or wound cover that causes wound healing by stimulating the patient's own tissue to regenerate.
In order to provide effective treatment for non-healing ulcers, the inventors of the present invention, have developed a temporary dermal wound dressing intended for use in the treatment of non-healing ulcers not limited to diabetic ulcers of the skin. It consists of a three-dimensional multi-layered tissue-like sheet of neonatal human dermal fibroblasts, mounted on one side of a porous chitosan sponge. The rationale for developing this product is that cells in a three-dimensional configuration are believed to be in a state closer to in vivo than cells in two dimensions, with better functions (Sakai Y, Furukawa K, Suzuki M. 1992. Immobilization and long-term albumin secretion of hepatocyte spheroids rapidly formed by rotational tissue culture methods. Biotechnol Tech 6:527-532.)
In the emerging field of tissue engineering, there is a requirement for developing tissue equivalents for both in vivo and in vitro uses. In some tissue equivalents that have been developed, there is significant contraction of the equivalent from its original size (Clark et al, 1989, J.Clin.Invest.84:1036-1040; Montesano et al, 1988, Proc. Natl. Acad. Sci. USA 85:4894-4897), which limits the usefulness of such equivalents to only specific applications in which the small contracted construct can be useful.
The tissue-like organization and constructs developed by the present inventor earlier by the novel method of macromass culture (Indian patent no. 195953 and US patent no. 20040082063), also are an example belonging to this contracting class - they spontaneously reduce in size over a period of time. Another example of cellular sheet which contracts when detached without support from the culture vessel is the multilayered sheet of keratinocytes (Green H, Kehinde O and Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci 1979; 76:5665-5668.).
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Therefore, there is a need to develop tissue equivalents which are non-contractile and methods that can render contracting tissue equivalents non-contractile to widen their usefulness and applications. For clinical use, a contracted graft of reduced size would not be useful. A graft with controlled specific size and in which the fragile cell sheet is supported for better handling would be more useful. One method to achieve non-contractility can be the use of a supporting scaffold or matrix on which the cellular sheet can be adhered so that it does not contract.
Cellular sheets have been cultivated over different supporting layers (Khor HL et .al. Preliminary study of a polycaprolactone membrane utilized as epidermal substrate. J Mater Sci Mater Med. 2003 Feb; 14(2): 113-20 and Imaizumi F et.al. Cultured mucosal cell sheet with a double layer of keratinocytes and fibroblasts on a collagen membrane Tissue Eng. 2004 May-Jun;10(5-6):657-64) some of which are non-porous sheets. Non-porous supports limit the supply and diffusion of nutrients and gases. Therefore, it is required that methods be developed which allow culture of cellular sheets or cells over porous scaffolds or matrices.. An inherent problem of culturing cells using porous matrices is that a proportion of total cells seeded onto the sponge in the form of cell suspension; leak out from the bottom of the sponge onto the base of the culture vessel. This problem has been recognized in earlier work in the field of cell culture methods (Yang J, Shi G, Bei J, Wang S, Cao Y, Shang Q, Yang G, Wang W (2002) Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid)(70/30) cell scaffolds for human skin fibroblast cell culture. J. Biomed. Mater. Res. 62(3)438-446.). This amounts to loss of cells when seeding, the number of cells remaining on the sponge being actually lesser than total cells seeded. This would result in wastage of cells, which is especially critical for tissue engineering applications wherein cell sources can be rather limited. It would also result in difficulty in getting reproducible constructs from equally seeded sponges, since variable numbers of cells would remain on the sponges, varying numbers being lost while seeding. Methods such as anhydrous ammonia plasma treatment and ethanol treatment have been used for preventing cell loss.
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Hence, for the formation of a cellular sheet over a porous matrix, there is a requirement for a method to be developed for preventing cell loss from the porous matrix when seeding. This would mean that the method should allow containment of cells on a side of the porous matrix for a suitable period of time. The present invention provides a cell friendly method that prevents cell loss through the pores of porous matrices when cells are seeded over the porous matrix to form a sheet, on one side of the porous matrix, thereby obviating the drawbacks associated with the conventional methods of preparation of cellular sheet over porous matrix.
In U.S. Patent no. 5,273,900. an epidermal cellular sheet has been made on one side of a porous collagen dermal substrate, which has been prepared by making a non-porous collagen-laminating layer on one side of the porous dermal component to be able to form the epidermal sheet. The laminating layer does not and is not intended to enter into the porous dermal component, and it remains an integral part of the final product. However, the present invention focuses on a gelatin-blocking component, which is not intended to form a laminating layer on one side, but to enter and fill the pores of the chitosan sponge, and later not remain a part of the final non-contracting tissue equivalent.
In another earlier invention (Pykett et al, U.S. Patent Application 20020197239 and U.S. Patent Application 20030096404.), involving methods for long-term culture of hematopoietic progenitor cells, the pores of a matrix are filled with a "gelatinous" substance. However the gelatinous substance of this invention holds the cells within itself in the pores of the matrix, and there is no sheet formation of the cells on one side of the matrix.
It is important that the tissue substitute remains viable during transportation without any specific need for cryopreservation, the present invention has specifically designed a system that addresses the needs of transporting a viable tissue substitute to the recipient's location.
The present invention provide a three dimensional tissue equivalent with high cell density comprising the dermal fibroblasts mounted on a porous matrix and the methods of preparation thereof, without any cell loss. The present invention has also focused on providing a tissue substitute, which can allow easy diffusion of growth factors from the cells
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into the wound bed by having direct contact of the cellular sheet with wound bed. In addition to the above the present invention, attempts are being made on producing the product under the invention in a cost effective manner so that the product under the invention can be easily be used by all sections of the society.
OBJECTS OF THE INVENTION
It is the object of the present invention to provide a three dimensional tissue equivalent with high cell density comprising a cellular sheet of dermal fibroblasts mounted on a porous matrix and the methods of preparation thereof without any cell loss.
It is the object of the present invention to provide a non-contractile three-dimensional tissue equivalent consisting of a macromass cellular sheet adhered on one side of a porous scaffold.
It is the object of the present invention to provide a three-dimensional tissue equivalent which is a dermal wound dressing.
It is the object of the present invention to provide a three-dimensional tissue equivalent, which expresses or produces enhanced amount of vascular endothelial growth factor thus causing local angiogenesis in the wound bed.
It is the object of the present invention to provide a three dimensional tissue equivalent which further produces enhanced amount of Interleukin-8 which aids in improving the clearance of bacteria by recruiting neutrophils to the wound site thus resulting in wound healing efficacy.
It is the object of the present invention to provide a three-dimensional tissue equivalent which comprises a matrix for the delivery of the cells, the thickness of such matrix does not impede diffusion of the growth factors.
It is the object of the present invention to provide a three-dimensional tissue equivalent which will enable the direct contact of the cell sheet with the wound bed.
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It is the object of the present invention to provide a three-dimensional tissue equivalent, which is flexible so that it can adhere at any site of the wound bed not limited to diabetic ulcers.
It is the object of the present invention to provide a three-dimensional tissue equivalent which can hold moisture thus aiding in the healing process.
It is the object of the present invention to provide a three-dimensional tissue equivalent that allows gas exchange so it prevents the build of toxic or unhealthy gases within the wound and also promotes natural healing.
It is the object of the present invention to provide a three-dimensional tissue equivalent for in-vitro screening of chemical for their toxicity.
It is the object of the present invention to provide a process of preparing a three dimensional non-contractile tissue equivalent by culturing a cellular sheet obtained by macromass culture technique over a porous scaffold or matrix.
It is the object of the present invention to provide a process of preparing a three-dimensional tissue equivalent, which prevents the loss of cells from porous matrices such as sponges when seeding.
It is the object of the present invention to provide a cell friendly method of preparing a tissue-equivalent consisting of a cellular sheet over a porous sponge, while preventing cell loss.
It is the object of the present invention to provide a three dimensional tissue equivalent comprising a multilayered tissue like sheet of neonatal human dermal fibroblasts mounted on one side of a porous chitosan sponge.
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It is the object of the present invention to provide a three dimensional tissue equivalent which enhances the rate of dermal regeneration as it comprises cells in a high cell density configuration which helps in accelerated wound healing.
It is the object of the present invention to provide three dimensional tissue equivalent comprising cells with high viability.
It is the object of the present invention to provide a three-dimensional tissue equivalent that does not require cryopreservation and hence is easier to transport.
SUMMARY OF THE INVENTION
The present invention provides a non-contractile three dimensional tissue equivalent with high cell density comprising a multilayered cellular sheet of dermal fibroblasts mounted on a porous matrix and the methods of preparation thereof without any cell loss.
The present invention also relates to the preparation and use of the three-dimensional tissue equivalent for wound dressing intended for use in the treatment of non-healing ulcers not limited to diabetic ulcers of the skin..
In one embodiment the present invention relates to three-dimensional tissue equivalent which consists of neonatal human dermal fibroblasts in a three-dimensional sheet configuration, mounted on one side of a porous chitosan biopolymer sponge disc. Each three-dimensional tissue equivalent is a circular flexible disc of diameter 3.0 cm and contains about 25 x 106 viable neonatal human dermal fibroblasts.
In one specific embodiment, the present invention relates to the culturing of cells using porous three-dimensional matrices having a sponge or foam structure without cell loss through the pores of porous matrices when seeding over the porous matrix with cells.
In one embodiment the present invention provides the three dimensional tissue equivalent which has enhanced expression of vascular endothelial growth factor (VEGF) and
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Interleukin-8 (IL-8). The high levels of VEGF are expected to induce angiogenesis in the wound bed. The high levels of IL-8 are expected to improve clearance of bacteria by recruiting neutrophils to the wound site. Local therapeutic angiogenesis, by delivery of angiogenic growth factors, is considered to be a promising approach in the treatment of ulcers associated with ischemia or peripheral arterial disease [Di Stefano R, Limbruno U, Barone D, Balbarini A. 2004. Therapeutic Angiogenesis of Critical Lower Limb Ischemia. Ital Heart J. 5 : 1-13.]. Another factor believed to contribute to the non-healing condition is chronic bacterial colonization. Interleukin-8 also has been shown to improve wound healing efficacy (Feugate et. al., 2002). In one preferred embodiment a single three dimensional tissue equivalent of the present invention secretes about 40 ng of VEGF and about 450-1000 ng of IL-8 in 24 hours after opening the package, under in vitro conditions. The tissue-like sheet also expresses other growth factors and extracellular matrix proteins involved in wound healing viz. transforming growth factors a and (3, basic fibroblast growth factor, platelet derived growth factor, collagen type I and III, fibronectin, syndecan 2 [Rosenberg L, Torre J de la. 2005. Wound healing, Growth factors. EMedicine.com,Inc]
In one embodiment the present invention provides a three dimensional tissue equivalent which provides cells in a high cell density configuration and thus would aid in accelerated wound healing, also due to the direct contact of the cell sheet with the wound bed.
In one embodiment the present invention provides a three dimensional tissue equivalent which provides cells that are capable of migrating, given a suitable substratum( such as wound bed), and further proliferating on the substratum.
In one preferred embodiment the present invention provides a three dimensional tissue equivalent wherein the base matrix is a chitosan (a natural biocompatible polymer) sponge which is highly porous, so will allow efficient gaseous exchange, which is a factor important for enhanced wound healing. The biopolymer sponge is also hydrophilic and its porous nature will help in maintaining a moist wound environment, which also promotes wound healing[Bryan J. 2004. Moist wound healing: A concept that changed our practice. J. Wound Care 13 :227-228.].
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In one embodiment the present invention provides a three-dimensional tissue equivalent, which is very flexible and is expected to fall in shape with the contours of the wound, improving contact with the secreted growth factors. The tissue equivalent of the present invention is not fragile, and is easy to handle with forceps, and provides visualization of the tissue sheet.
In one embodiment the present invention provides a three dimensional tissue equivalent which does not allow the accumulation of the exudates produced by the wound as it enables the exudates to ooze out from the edges of the porous sponge disc, where the tissue sheet is not present ( about 2-3 mm along the edge of the circular sponge base).
In one embodiment the present invention provides transportation of the three-dimensional tissue equivalent in a individually aseptically packed in a especially designed sterile square pouch, which contains 2.5 ml sterile transport medium. The pouch is contained in an outer square plastic cassette by which sufficient portion of tissue substitute with high cell remains viable during transportation without any specific need for cryopreservation and can be transported to the recipient's location.
The main features of the three-dimensional tissue equivalent of the present invention are as follows:
1. It provides cells in a three-dimensional configuration, supported by a matrix that is
porous.
2. It provides cells at a high cell density.
3. It provides cells with high percentage of viability.

4. It can express or produce enhanced amount of vascular endothelial growth factor and IL-8 and allows its easy diffusion to the wound bed, which aids in healing of ulcers.
5. It enables direct contact of the cells with the wound bed, with no matrix between the cell sheet and the wound bed, since the cell sheet is formed entirely on one side of the matrix.
6. It enables transfer of cells to the substratum such as wound bed.
7. It is flexible enabling it to adhere to the any site of wound especially foot.
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8. It has a porous scaffold, so can hold moisture thus providing a conducive environment for
faster healing.
9. It has a porous scaffold, allows gas exchange, which aids in preventing the build-up of
gases in the wound bed.
10. It does not allow any accumulation of the exudates from the wound.
11. It does not require cryopreservation thus reducing the transport costs.
12. It provides cells, which are viable upto 72 hours.
13. It provides a cost effective tissue substitute.
14. It is safe and is efficient in treating wound ulcers.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form the part of the present invention and are included to substantiate and demonstrate the important aspects of the disclosure. The present invention may be better understood by the following drawings in combination with the detailed description of the specific embodiments presented herein.
Figure 1: (A) Illustrates the top view of the three dimensional tissue equivalent mounted on one side of a porous chitosan sponge disc which is aseptically packed in a sterile square biocompatible pouch containing sterile transport medium. The pouch is then contained in an outer square plastic cassette. (B) Illustrates the tissue equivalent after incubation in MTT solution, with the cellular sheet turned dark on the white sponge.
Figure 2 depicts cell loss through a chitosan sponge when seeding. A representative microscopic view of the base of the culture dish in which the chitosan sponge was placed for seeding is depicted, after the sponge was removed from the dish. Culture dish in which chitosan sponge was not filled with gelatin solution, hence pores were not blocked, showing large number of cells leaked out. There were no cells lost from the sponge that was filled with gelatin.
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Figure 3: Illustrates the In vitro Tumorigenicity results of soft agar assay of cells isolated from the tissue equivalent of the present invention wherein cell line B16 melanoma was a positive control.
Figure 4: Illustrates a representative normal karyogram of cells isolated from the tissue-equivalent of the present invention.
Figure 5: Illustrates the analysis of the extracted fibroblasts from the tissue equivalent of the present invention for expression of HLA-DR surface protein by Flurorescence activated cell sorting (FACS).
Figure 6: Illustrates the Histology showing the haematoxylin and eosin staining of vertical section through the tissue equivalent of the present invention, demonstrating the multi-layered organization of cells, at a high density, which would contribute to accelerated wound healing. It also shows the highly porous nature of the supporting matrix. (A) High power view. (B) Low power view.
Figure 7: Illustrates the expression of genes involved in wound healing by the cells of the tissue equivalent of the present invention.
Figure 8: Illustrates the comparison of expression of genes between tissue-equivalent of the present invention and fibroblast monolayers, wherein 18S rRNA expression serves as the control, the level of which is unchanged.
Figure 9: Illustrates the transfer of cells to wound bed from the tissue equivalent (substratum) after 24 hours.
Figure 10: Illustrates the SDS page analysis of the residual protein of the transport medium used for the tissue equivalent of the present invention.
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Figure 11: Illustrates the preclinical study of efficacy and safety of the three dimensional tissue equivalent of the present invention in a wound healing animal model wherein selected photographs of the histological sections (haematoxylin & eosin staining) through wound healing area at different time points are depicted.
Figure 12: Illustrates the result of the test for cytotoxicity to show that the tissue equivalent of the present invention is not cytotoxic to fibroblasts.
Figure 13: Illustrates the in vitro use of the three dimensional tissue equivalent of the present invention in toxicity testing of chemicals.
DESCRIPTION OF THE INVENTION
Definitions:
The term "three dimensional tissue equivalent" as used herein refers to a three-dimensional arrangement of dermal fibroblasts to form tissue like construct and a three-dimensional structure of matrix.
The term "cell loss" as used herein refers to the number of cells not incorporated into the tissue-like sheet and that it is lost during the seeding of the cells to form a tissue-like sheet.
The term "non contractile" as used herein refers to a tissue equivalent wherein the edges of the tissue-like sheet, do not contract when placed on a substrate and that it retains its initial size and shape in the stretched form.
The term "high cell density" as used herein refers to a certain high seeding density of cells within a favorable range is required to be achieved within a given space. In the macromass range of in a range of lx l06 to l0x 106per cm2 high cell seeding densities, when the cells are settled together within the three-dimensional space that is occupied by the cells at the base of the culture vessel, they come into a state of close proximity with one another that triggers or signals them into a tissue formation mode by which they become cohesively integrated.
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The term " macromass culture" as used herein refers to the formation of macroscopic three-dimensional tissue-like constructs, wherein "macroscopic" means that the size of the tissue is at least such that it can be easily visually discerned by the normal unaided human eye and a culture system for three-dimensional tissue-like formation or organization of cells, in which, cells are seeded at a high density per unit area or space of a culture vessel and there is no requirement for any other agents that aid in tissue formation. A broader definition of macromass culture is a method of generating three-dimensional tissue-like organization, macroscopic or microscopic, from cells by high-density cell seeding, bringing cells together in close proximity in a certain favorable range of high densities of cells in three-dimensional space, that favors cohesive integration of cells into a three-dimensional tissue-like state, there being no requirement for any other agents that aid in tissue formation.
The present invention describes development of a non-contractile tissue-equivalent, which is a dermal wound dressing, based on the tissue-like constructs made by macromass culture as described in detail in Indian patent number 195953, which are inherently contractile. The parent application has covered the macromass culture technique for the formation of tissue like constructs. In Indian patent number 195953 the invention has demonstrated the preparation where in the tissue like construct formed was contractile in nature.
The present invention is an advancement to the Indian patent number 195953 and a novel method is developed under the present invention, which helps in obtaining non-contractile macromass tissue-like constructs.
The present invention has focused on the delivery of the non-contractile tissue like construct wherein the tissue construct gets adhered to a support while ensuring that no cells are lost during formation. The present invention has developed method for delivering the macromass tissue like construct in a non-contractile form in that it is held to a support in its original size. In order to achieve the adherence of the tissue like construct to a support, a support with q rough surface or support is required, which should be porous, to allow the exchange of nutrient and gas. Therefore the present invention has selected a porous support with a rough surface as this has proved to be more effective in producing the desired result of the
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invention as the tissue sheet does not adhere to a smooth surface. In view of this requirement, the present invention has selected a porous sponge or matrix whose surface is not smooth due to the microscopic projections.
Since the best option was to select a porous rough surface for support it was a challenge for the inventors to prevent the cell loss from porous support. Further to this, a methodology was devised under the present invention, as detailed herein the specification. By this methodology, the macromass cellular sheet was effectively cultured over the surface of a chitosan sponge to ensure that conditions are maintained that prevents the leaking of cells form the tissue like sheet developed by the present invention. The tissue-like sheet was formed entirely on one side of the sponge and adhered well without coming off even with prolonged incubation, thus rendering it non-contractile without reduction in its original size throughout.
The present invention further describes a method for seeding of porous three-dimensional matrices or biodegradable or non-degradable porous polymer scaffolding matrix having a sponge or foam structure without cell loss through the pores of porous matrices when seeding the porous matrix with cells. To our knowledge till date there has been no public report of such a method of preventing cell loss from porous matrices such as sponges or foam when seeded.
According to the method of the present invention, the porous matrix having a sponge or foam structure, prior to seeding of cells, is placed in a culture dish containing a molten solution of a substance, gelatin, such that the molten solution is absorbed into the pores of the sponge. Suitable conditions are maintained so that the molten solution solidifies or sets, within the pores of the sponge. The cells are then seeded onto the porous matrix having a sponge or foam structure and allowed to attach to the upper surface of the sponge or foam, under conditions in which the solution of blocking substance remains solidified. Tissue-like organization of cells by macromass method takes place during this time, enough to result in cohesion of cells such that the cells are not free any longer, but attached to each other and the top surface of the sponge. After allowing formation of the macromass sheet, the conditions of
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incubation of the assembly are changed to one that causes the solution of gelatin to liquefy and drain out of the pores of the sponge or foam, into the culture medium in subsequent washings. The cells are already integrated into the macromass sheet, so they do not disperse through the pores even after the gelatin has liquefied.
Thus, in the present invention, the pores of a porous matrix are temporarily filled or blocked with a substance for the purpose of preventing cell loss, not allowing cells to leak through while seeding. This prevents cell loss. The blocking substance is later removed from the porous matrix, after cells have formed the macromass sheet on one side of the sponge, to regain porosity or 'openness' of pores of the sponge. In the present invention, cells are not intended to be entrapped within or impregnated into the substance that is used for blocking. The role of the blocking substance in the present invention is to block or prevent the entry and passage of cells into the porous matrix. The blocking substance is temporary in nature, and does not remain to be an integral part of the final tissue-equivalent.
The porous matrix used in the method of the present invention can be of varying pore size. The porous matrix used in the method of the present invention is formed of material selected from the group not limited to gelatin, chitosan, collagen, polyglycolic acid, polylactic acid, alginate. In the preferred aspects Chitosan is used selected as a porous matrix. Chitosan is a natural biocompatible polymer, which is obtained by the alkaline deacetylation of chitin, which is derived from the exoskeletons of crustaceans such as crabs (Shi et. al., 2006). Chitin is a co-polymer of N-acetyl-glucosamine and N-glucosamine linked by glycosidic bonds.
The blocking/filing substance used in the method of the present invention is selected from the group not limited to gelatin, alginate, pectin, agar, agarose.
In the Pykett invention (US patent application 20020197239 and US patent application 20030096404), the rationale for using a gelatinous substance is to impregnate the cells into it to provide attachment and a three-dimensional environment within the pores of the matrix. In the present invention, the rationale of using gelatin is to keep cells out of the porous matrix to allow them to form a sheet entirely on one side of the matrix. Secondly, in the Pykett
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invention, the gelatinous substance after achieving solidified state, remains in the final product, becoming an integral part of it - there is no reliquefication of the gelatinous substance. In the present invention, the gelatin is removed after cell sheet formation, by changing conditions to liquefy it. Thirdly, in the Pykett invention, the list of exemplary "gelatinous" substances actually does not include gelatin itself. This would be because feasible concentrations of gelatin cannot be maintained in a solid state at 37°C, which is the optimal incubation temperature for cells, and the gelatinous substance is required to be solid at 37 °C in that invention. While in the present invention, gelatin is used because a substance is required that would liquefy at 37 °C.
The present invention thus provides a three dimensional tissue equivalent which can be used as dermal dressing for various wounds not limited to diabetic ulcers, pressure ulcers and venous ulcers.
Additionally, the tissue-equivalent of the present invention has potential to be an in vitro model of normal non-dividing cells for safety testing. The macromass three-dimensional sheet which is mounted on a chitosan sponge in the present invention, contains the cells in a non-dividing state. Thus, these are normal cells in a non-dividing state, which can be a model of the normal quiescent cells of the human body, in contrast to the abnormal tumour cells which are rapidly dividing. Many of the anti-cancer drugs that are developed are based on the principle that they act on rapidly dividing cells (cancerous cells) and not on quiescent cells (normal cells of the body). This is important because the anti-cancer drug should not kill normal cells while destroying cancerous cells. Hence, in the development of anti-cancer drugs which act by destroying rapidly dividing cells, it is important to establish its safety towards normal cells. An in vitro model to establish this safety would be useful, since currently, it is preferred that safety testing in animals is minimized by the use of in vitro alternatives.
The following steps are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice.
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However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLES 1: PREPARATION OF THE THREE DIMENSIONAL TISSUE EQUIVALENT.
I. Cell isolation and culture.
In the present invention, human dermal fibroblasts were isolated from discarded human skin biopsies obtained with written informed consent. The dermis was separated from the epidermis by treatment with Dispase (Sigma, St. Louis, USA). The dermis was minced and digested with 0.01 % collagenase in DMEM + 10 % FCS overnight and then cells were allowed to attach. Cells were cultured in DMEM + 10 % FCS at 37 °C in 5% C02 and subcultured using Trypsin-EDTA solution.
II. Preparation of chitosan sponges.
Chitosan sponges of diameter 3.0 cm and thickness about 1.5 mm are prepared by lyophilization of frozen chitosan solution in 3.5 cm dishes. After lyophilization, the chitosan sponges are stabilized in isopropanol. The chitosan sponges are treated with ammonia and methanol solution. Chitosan sponges are rinsed with water for 3 hours with shaking. Chitosan sponges are equilibrated in isopropanol. Chitosan sponges are gamma-irradiated in pouches containing Vitamin E dissolved in isopropanol. Gamma-sterilized chitosan sponges are rinsed in isopropanol. Chitosan sponges are soaked in serum-free medium overnight at 37°C.
III. Preparation of Non contractile three dimensional tissue substitute
The cell culture system used was that of generating tissue-like organization as described in Indian Patent number 195953 and US patent application 20040082063 in combination with a chitosan sponge by an inventive method, the latter being die novel aspect of the present invention. Thus, the aim was to construct a composite object consisting of a chitosan sponge with macromass tissue-like organization of dermal fibroblasts on one side.
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1.0 gm gelatin is dissolved in 10 ml Dulbecco's phosphate buffered saline by heating in a microwave oven. The gelatin solution is filter-sterilized through 0.2 um syringe filter while molten, and poured into 3.5 cm dishes, about 3.0 ml in one dish. Chitosan sponge, pre-soaked as above, is placed in the molten gelatin solution and pressed with forceps so that the gelatin enters the pores and sponge pores are completely filled with gelatin solution. The gelatin-soaked chitosan sponge is transferred to another 3.5 cm dish, which is then placed in the refrigerator to set the gelatin within the sponge. Dermal fibroblasts are harvested from culture flasks and collected in a tube in growth medium. The cells are counted in a cell counting chamber. Volume of cell suspension containing 25 x 106 total cells is transferred to a fresh tube, for a single tissue equivalent of the present invention. The cells are pelleted at 1000 rpm for 5 minutes and resuspended in 2.0 ml medium with 10% fetal bovine serum. The chitosan sponge is removed from the refrigerator and brought to room temperature for 5 minutes. The excess set gelatin from the top of the sponge is scraped away by gently scraping using a cell scraper. A sterile stainless steel ring of outer diameter 3.3 cm and inner diameter 2.5 cm and thickness or height of 0.4 cm is placed over the sponge.
The 2.0 ml cell suspension containing 25 x 106 cells is seeded over the sponge, within the
stainless steel ring. This gives a seeding density of 5 x 10 cells per cm , since the area within the ring is 5 cm . The dish is carefully placed in an incubator at 28-29°C for 2 hours and 15 minutes. The gelatin is semi-solid at this temperature. The dish is carefully transferred to a 37°C CO2 incubator and incubated for 2 hours and 15 minutes. The gelatin liquefies at this temperature. The dish is removed from the incubator and the stainless steel ring is removed by lifting. The tissue equivalent of the present invention is carefully lifted using forceps, holding at the edge and placed in a dish containing 25 ml growth medium. The tissue equivalent of the present invention is incubated at 37 °C in an CO2 incubator overnight, which allows full formation of the macromass cell sheet adhered to the chitosan sponge, and allows the gelatin to leach out of the sponge into the medium, causing removal of gelatin. The tissue equivalent of the present invention is transferred to a fresh dish containing fresh growth medium. Then it is packaged in transport medium in a pouch ( FIGURE 1A). To better visualize the macromass cellular sheet on the chitosan sponge, the tissue equivalent was
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incubated in MTT solution so that the cells, being viable, formed a dark purple colour. (Figure IB).
Evaluation of cell loss through sponge.
The 3.5 cm dish in which seeding was done was assessed for cell loss by viewing the dish under the microscope for presence of cells attached to the base of the dish. It was found that substantial number of cells had been lost from the sponge that had been seeded with same number of cells, but had not been filled with gelatin solution. There was no or negligible cell loss from the sponge filled with gelatin before seeding. The results are depicted in FIG.2 Thus, in this embodiment of the present invention; cell loss from a chitosan sponge was successfully prevented by using the method of this invention, and as can be seen from the method used to prevent cell loss as described above, only cell-friendly agents like gelatin and phosphate buffered saline are used.
EXAMPLE 2: CHARACTERISATION AND EVALUATION OF THREE DIMENSIONAL TISSUE EQUIVALENT.
The three dimensional tissue equivalent of the present invention was evaluated for safety and effectiveness and the data are classified below in four categories (1) Safety (2) Potency (3) Purity (4) Stability.
1. Safety
A) Sterility : To ensure that the aseptic conditions were maintained from the manufacturing process till final packing, the batches of tissue equivalent of the present invention were tested for sterility to detect the presence of aerobic and anaerobic microbes. This test is performed by Direct Inoculation method (IP 1996), which involves inoculating the test sample in two different sterile nutritive media, namely, Fluid Thioglycollate Medium (FTM) and Soybean Casein Digest Medium (SCDM). Absence of growth in the inoculated media during the incubation period of 14 days confirmed the sterility of the samples.
B) Bioburden : The microbial load in terms of number of colonies appearing on plates of solid medium was checked as index of the microbial density or bioburden entirely during the
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manufacturing process and the packed product. The final spent transport medium was also tested for microbial burden.
C) In vitro Tumorigenicity.
The tumorigenicity of the three dimensional tissue equivalent of the present invention was tested by soft agar assay. In this assay, tumorigenic cells, which are not dependent on attachment, form colonies, while normal cells which are dependent on attachment, do not form colonies. While the positive control cell line B16 melanoma, formed colonies within 28 days of incubation, the cells from tissue equivalent of the present invention did not form colonies and remained as single cells, thus confirming that the cells are non-tumorigenic and the process does not induce tumorigenicity. A representative result is depicted in Figure 3
D) Karyology.
The chromosomal abnormalities of the tissue equivalent of the present invention was analysed by karyotyping wherein the dermal fibroblast were extracted from the tissue equivalent and plated and then karyotyped. The test confirmed that the cells have a normal karyotype with no detectable chromosomal abnormalities as shown in the figure 4.
E) Expression of HLA-DR surface protein.
As HLA-DR molecule on the surface of cells is principally responsible for the immune rejection of allogeneic cells, the banked cells used for preparing the tissue equivalent of the present invention are confirmed to be at least 98% negative for HLA-DR surface protein expression, by culturing till passage 7 or more. In order to ensure that the process of preparing tissue equivalent of the present invention from these banked cells does not further enhance the surface expression of HLA-DR protein, the fibroblasts were extracted out from the prepared tissue equivalent of the present invention. The extracted fibroblasts were analyzed for HLA-DR surface protein by fluorescence activated cell sorting (FACS). It was found that the extracted cells extracted did not have enhanced expression of HLA-DR surface protein compared to the banked cell monolayers. The extracted cells were also minimum 98% negative for HLA-DR. A representative result is shown in Figure 5.
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2. Potency
A) Histology
Three-dimensional organization of cells is the key rationale for developing the tissue equivalent of the present invention. As described in the foregoing, the three-dimensional organization of the fibroblasts is expected to give better results in wound healing (than monolayer of cells). Therefore, to confirm the three-dimensional nature of the tissue equivalent of the present invention, histological examination was performed. Figure 6 shows the haematoxylin and eosin staining of vertical section through the tissue equivalent, demonstrating the multi-layered organization of cells, which can be also seen to be at a high density. Figure 6A shows the high-power view, and Figure 6B shows the low power view wherein both top and lower sides of the chitosan sponge are encompassed in the picture.
It can be seen from the figure that the cellular sheet is entirely on one side of the sponge. Figure 6 also shows the highly porous nature of the chitosan scaffold, which is also an important attribute in the mechanism of action of the tissue equivalent of the present invention, since the porous nature would allow gas exchange and help in maintaining a moist wound environment. Also, it can be seen from the figure that the cellular sheet on one side of the sponge would be in direct contact with the wound, thus there would be efficient diffusion of growth factors from tissue equivalent to the wound bed, there being no matrix impeding the diffusion. It can also be seen from the figure that the pores of the sponge are 'open', in that they are devoid of integrated matter filling or occupying them. This is in contrast to other applications of porous scaffolds, wherein the pores become occupied with a filling material and/or cells and extracellular matrix synthesized by the cells so that the pores are no longer 'open' in the final construct.
B) Viability of cells.
For the tissue equivalent of the present invention to be efficacious, it is important that the viability of the cells in the tissue equivalent, after preparation, is high, and the end-result of the process gives a product with high cell viability. The cells were extracted out from the tissue equivalent by trypsinization. The viability of the extracted single cell suspension was checked using the vital dye Trypan Blue, and counted using a counting chamber. It was
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found that cells in the prepared tissue equivalent had a viability of at least 95%, which contributes to the high efficacy of the product. This high viability also indicates that the process used to prepare the tissue-equivalent without cell loss is cell-friendly.
C) Expression of genes involved in wound healing.
The expression of the genes for transforming growth factor pi (TGFb1), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), transforming growth factor a (TGFα), platelet derived growth factor (PDGF), collagen type I, and collagen type III, each of which has an important role to play in the wound healing process of the skin, was checked by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) method. This method detects the messenger RNA transcribed from expressed genes. The data are shown in Figure 7. Since these genes were expressed by the tissue-equivalent, this demonstrates the capability of the three dimensional tissue equivalent of the present invention to effect wound repair.
In addition, a comparison of the gene expression between monolayers of dermal fibroblasts and tissue equivalent of the present invention was done by RT-PCR. It was found that the tissue equivalent of the present invention has greatly enhanced levels of vascular endothelial growth factor (VEGF) and interleukin-8 compared to monolayers. Thus, the tissue equivalent of the present invention presents dermal fibroblasts in a more desirable phenotype than simple cultured monolayers, with respect to ability to induce angiogenesis mediated by VEGF in the wound bed, and recruitment of neutrophils, mediated by Interleukin-8, to the wound bed, which would aid in clearance of bacterial colonization and enhance wound closure efficacy. The data are shown in Figure 8. 18S rRNA expression serves as the control, the level of which is unchanged.
VEGF is produced by cells in different isoforms, which result in VEGF proteins of different sizes and different functionality, namely VEGF206, VEGF 189, VEGF 165 and VEGF121. Of the VEGF forms, VEGF 165 is the one that has optimal characteristics of bioavailability and biological potency (Ferrara, et. al., 2003 The biology of VEGF and its receptors. Nature Med. 9(6)669-676.).. Thus, in order to ensure that the major VEGF isoform produced by the tissue equivalent, which is enhanced as shown in the figure, corresponds to VEGF 165, the
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RT-PCR product was sequenced. The sequence was compared to the known sequence of VEGF165, and this confirmed that the sequence indeed was of VEGF165.
D) Secretion of vascular endothelial growth factor.
The biological activity of a gene is carried out by the protein, which is translated from the messenger RNA transcribed from the gene. Thus, it is the protein, which is responsible for the biological activity. VEGF165 protein has the activity of inducing angiogenesis, and for its function, it should be secreted from the cells. In order to establish that VEGF165 is secreted out from the cells of the tissue equivalent, and to quantify the amount of secreted VEGF165, the tissue equivalent of the present invention were incubated at 37°C in culture medium for 24 hours. Then the culture medium was collected and the VEGF165 in it was detected and quantified by Enzyme Linked Immunosorbent Assay (ELISA) using antibody specific for the VEGF165 protein form. The culture medium was positive for secreted VEGF165 and by quantification, it was found that a single tissue equivalent of the present invention produced about 40 ng of VEGF165 in 24 hours after opening the package, under in vitro conditions. This amount of VEGF165 corresponds to biologically potent levels (Mansbridge et. al., 1999 Growth factors secreted by fibroblasts : role in healing diabetic foot ulcers. Diabetes Obes. Metab. 1(5)265-279.).
E) Secretion of Interleukin-8.
As described above for VEGF165, Interleukin-8 protein also exerts its action after secretion from the cells. In order to establish that secreted Interleukin-8 is produced by the tissue equivalent of the present invention and to quantify the amount, the tissue equivalent were incubated at 37°C for 24 hours in culture medium. The culture medium was collected and secreted Interleukin-8 in it was detected and quantified by ELISA. The test was positive for secreted Interleukin-8 and it was found that a single tissue equivalent produced about 450 to 1000 ng of Interleukin-8 in 24 hours after opening the package, under in vitro conditions. This amount of Interleukin-8 corresponds to biologically potent levels (Martin et. al., 2003 Effect of human fibroblast-derived dermis on expansion of tissue from venous leg ulcers. Wound Rep. Reg. 11(4)292-296.)
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F) Transfer of cells to substratum.
In order to test the migration of the cells from the three dimensional tissue equivalent onto a substratum, the tissue equivalents were inverted and incubated on tissue culture plates in growth medium. It was found that, after 24 hours of incubation, cells had migrated to the tissue culture plates, which is shown in Figure 9. This would contribute to the efficacy of the tissue equivalent in wound healing.
3. Purity.
A) Endotoxin :
Presence of bacterial endotoxins obtained from the cell wall of gram-negative bacteria is responsible for inducing high temperatures in humans. To ensure that the manufacturing process and the product have endotoxin below the acceptable limit, the packages were aseptically opened and the final spent transport medium was tested for endotoxin. And was determined by the gel-clot technique using the Limulus Amoebocyte Lysate (LAL) reagent. When incubated at 37°C for one hour in the presence of bacterial endotoxins, the LAL reagent forms a firm gel-clot. Failure to form a gel-clot under the conditions of the test indicates absence of detectable endotoxin in the sample. The spent transport medium of the final packaged tissue equivalent of the present invention was tested to have endotoxin level B) Residual protein in the final product.
To evaluate the level of protein in the transport medium and the washes, the tissue equivalent of the present invention were prepared, packaged, opened and rinsed in saline. The spent transport medium and washes were collected and analyzed for presence of protein by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining, which is a highly sensitive method of detecting protein in gels. The result from three batches of three-dimensional tissue equivalent is shown in Figure 10. As can be seen, there is only a trace amount of protein in the spent transport medium, which is carried over from the upstream process, while in the first wash itself, there is no trace of any protein.
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4. Stability.
A) Effect of transport conditions on viability.
The tissue equivalent of the present invention is stored and transported in a hypothermic storage transport medium, at 2-8°C. To ensure that viability is maintained at 2-8°C, batches of the tissue equivalent of the present invention were prepared, packaged, and kept at 2-8°C for a period of 72 hours. Then the packages were opened, the tissue equivalents were rinsed with saline, and then the cells were extracted out of them. The viability of the cells was assessed using Trypan Blue vital dye and a counting chamber. Additionally, the pH of the transport medium in which the tissue equivalent of the present invention are packaged is an indication of the metabolic state of the cells while in transit. In hypothermic storage, in order that the cells remain viable, they should not metabolize and thus the medium pH should not change. The pH would be affected if the cells had metabolized or the state of the cells was adversely altered. Therefore, in this study, after opening the packages, the pH of the spent transport medium was checked. It was established that :-(i) The tissue equivalent had a viability of at least 97% after 72 hours storage at 2-8°C,
which is a very high viability, (ii) The pH of the spent transport medium was about 7.0, which indicates that the state of the
cells is not adversely affected upon hypothermic storage at 2-8°C for 72 hours.
B) Effect of the transport conditions on the integrity
An important aspect of stability of product is that the product should maintain mechanical integrity under conditions of transport and handling, during which there is agitation and impact. Therefore, a simulated study was carried out, in which batches of the tissue equivalent were placed in a transport box and then subjected to continuous agitation for two periods of about 2 hours, and also subjected to impact. Then the box was opened and the tissue equivalents were assessed for maintenance of mechanical integrity. It was established that there was no loss of integrity of the tissue equivalent, and no alteration in shape.
EXAMPLE 3: IN VIVO STUDIES AND TOXICOLOGY
1. Study of efficacy and safety of tissue equivalent of the present invention in a wound
healing animal model.
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The efficacy and safety of the tissue equivalent of the present invention was assessed by performing a skin wound healing study in SCID CB17 mice. SCID mice are severe-combined-immuno-deficient mice, which were used since the dermal dressing to be tested consists of human cells, which would undergo xenograft rejection if tested on animals that are non-immuno-compromised. The study was carried out n accordance with CPCSEA guidelines and with IAEC approval. There were four sets of animals, for different periods of time after surgery and application, namely, 4 days, 8 days, 12 days, and 16 days. For each time point, there were three Control animals and six animals treated with the tissue equivalent. In the "Control" group of animals, chitosan sponge alone was applied on each wound. In the "Treated" group of animals, the tissue equivalent was applied on each wound, with the cellular sheet side facing the wound bed. Animals were anaesthetized and surgery was performed in a laminar flow workstation. A single full-thickness wound of 6 mm diameter was created on the back of each animal, using a punch biopsy instrument. After application of tissue equivalent or chitosan sponge alone, the wounds were dressed with bandage. The animals were observed for the above mentioned time points. At the end of each time point, animals were sacrificed and skin tissue was obtained from the wound healing area of each animal. The tissues were fixed and analyzed histologically with respect to various parameters.
The main observations from the preclinical evaluation were :-(i) Faster rate of complete epithelization in treated animals compared to Control animals, (ii) Earlier angiogenic response or neovascularization in treated animals compared to Control
animals, (iii) Earlier formation of new extracellular matrix (ECM) in treated animals compared to
Control animals, (iv) Enhanced accumulation of polymorphonuclear leucocytes (PMN) in treated animals
compared to Control animals, (v) There was no foreign body reaction (foreign body giant cells) in either treated or Control
animals, (vi) The chitosan sponge was not integrated into the skin after complete wound closure, either
in treated or Control animals.
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(vii) There were no adverse events seen in either treated or Control animals, such as, oedema, erythema, or fluid collection.
Details of the data are summarized in Table 1, and selected photographs of the histological sections (haematoxylin & eosin staining) through wound healing area at different time points are depicted in Figure 12. Thus the studies proves that application of the tissue equivalent of the present invention enhanced the healing of full-thickness wounds in SCID mice, compared to control animals, and has shown no adverse events of application. Table 1.

Parameter/ Timepoint 4 days 8 days 12 days 16 days
Control Treated with tissue equivalent Control Treated with tissue equivalent Control Treated with tissue equivalent Control Treated with tissue equivalent
Neovascularization 0% 60% 66% 83% 100% 83% 100% 100%
PMN>10per 20x field 100% 100% 100% 100% 0% 66% 0% 0%
New ECM formation 0% 80% 100% 100% 100% 100% 100% 100%
Epithelization
3 mm or less gap 0% 20% 0% 66% 100% 100% 100% 100%
1 mm or less gap 0% 0% 0% 16% 0% 83% 100% 100%
0 mm gap 0% 0% 0% 0% 0% 50% 100% 100%

The values given for each parameter is the percentage of animals positive for that parameter
in each group. Percentage increase of 50% or greater in treated animals over Control animals is underlined. In the Epithelization parameter, there are 3 categories, viz., gap remaining for complete epithelization 3 mm or less, 1 mm or less, and 0 mm (ie complete epithelization). PMN = Polymorphonuclear leucocytes; ECM = Extracellular matrix.
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Foreign body reaction : Not seen in any animal.
Integration of sponge into skin : Not seen in any animal.
Other adverse events : Not seen in any animal (eg. oedema, erythema, fluid collection)
2. TOXICOLOGY
A) Safety of the three dimensional tissue equivalent.
As described earlier, the safety of the three dimensional tissue equivalent of the present invention upon application on full-thickness wounds in mice has been confirmed, wherein, there were no signs of reactivity (oedema, erythema, fluid collection) upon intracutaneous application in the full-thickness wounds created on the animals.
B) In Vivo Tumorigenicity of the three dimensional tissue equivalent.
The in vivo tumorigenic potential of cells from the three dimensional tissue equivalent of the present invention a dermal wound dressing was studied.with IAEC approval by injecting end-of-production stage fibroblasts extracted from the tissue equivalent into SCID mice. A total of lxl06 cells (98% cell viability), suspended in 50 ml of sterile normal saline, was injected (i.m.) in the left hind limb of each of six SCID CB17 mice. The mice were observed for three months.
There were no tumors developed and it was concluded that cells from the tissue equivalent of the present invention are non-tumorigenic.
C) Ames Mutagenicity :
In order to assess if the spent transport medium of the tissue equivalent has mutagenic property, Salmonella typhimurium Reverse Mutation Assay, Test no. 471. was conducted according to the OECD Principles of Good Laboratory Practice (1982). The tissue equivalent of the present invention were prepared, packaged, and kept at 2-8°C for 72 hours, then the packages were opened and the spent transport medium was collected and tested as per the above mentioned assay. The spent transport medium was tested at the concentrations of 61.72, 185.18, 555.55,1666.67 and 5000 ug/plate using sterile distilled water as solvent. The study was performed without and with metabolic activation (S9 fraction) prepared from sodium phenobarbital induced rat liver. The solvent control and appropriate positive controls
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were tested simultaneously. Plating was done in triplicate for each concentration of test substance. The study showed that the mean numbers of revertant colonies counted at different concentrations of test substance were comparable to that of the controls, in the absence and presence of metabolic activation. The number of revertant colonies in the positive controls increased by 3.93 to 95.33 fold under identical conditions.
Hence the spent transport medium tested at 61.72, 185.18, 555.55, 1666.67 and 5000 jig/plate did not induce mutations in Salmonella typhimurium upto the maximum concentration of 5000 |j.g/plate, and is not mutagenic in this Salmonella Reverse Mutation Assay.
D) Hemolysis of red blood cells : The spent transport medium of the tissue equivalent was
evaluated for hemolytic activity wherein it assessed for hemolysis using freshly collected
heparinized rabbit blood. Heparinized rabbit blood was added to each of negative control,
positive control, and undiluted test spent medium, each in triplicate. The samples were mixed
and incubated at 37°C for 1 hour. They were then centrifuged at 3500 rpm for 5 minutes. The
absorbance of the supernatants was measured at 545 nm, the percent hemolysis shown by the
spent transport medium of tissue equivalent of the present invention was 0.38% which is less
than the acceptable limit of 5%, hence it is non-hemolytic.
E) Cytotoxicity : The cytotoxicity of the tissue equivalent of the present invention was
studied wherein its effect on the growth of monolayers of dermal fibroblasts was taken as the
measure of cytotoxicity, since dermal fibroblasts are important cells in the wound bed of
non-healing ulcers, on which tissue equivalent of the present invention is to be applied.
Equal numbers of fibroblasts as monolayers were incubated by themselves or in the presence
of tissue equivalent of the present invention by placing it in cell culture inserts over the
monolayers. Growth of the fibroblast monolayers was assessed after 48 hours by MTT
staining, which was quantitated by measuring absorbance at 570 nm. The growth of
fibroblast monolayers in the absence of tissue equivalent of the present invention was taken
as 100%, ie, a fold change of 1.0 in growth. The result of the cytotoxicity assay is shown in
Figure 13. It was observed that the growth of fibroblast monolayers was not decreased or
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adversely affected when incubated in the presence of tissue equivalent of the present invention, compared to without it. There was an increase in growth of fibroblasts, when incubated in the presence of tissue equivalent of the present invention. Hence the tissue equivalent of the present invention was found to be non toxic towards cells namely dermal fibroblasts.
EXAMPLE 4: Use of the tissue-equivalent of the present invention for in vitro purposes:-
1. As an in vitro model for drug testing : As mentioned in the description, the tissue
equivalent of the present invention, can be an in vitro model of normal non-dividing cells, for
safety testing of anti-cancer compounds.
To test its usefulness as an in vitro model, dividing monolayers of fibroblasts and the tissue-equivalent of the present invention were incubated with or without (control) camptothecin, an anti-cancer drug which acts on dividing cells. Both were incubated for the same period of time. Then the metabolic activity was assessed by incubating both in MTT, which was quantitated by spectrophotometry. The figure shows the results. It was found that camptothecin was toxic to the actively dividing cells, while it showed no toxicity towards the non-dividing tissue-equivalent of the present invention. This confirmed the known property of camptothecin. Thus, it demonstrated that the tissue-equivalent of the present invention has potential for in vitro use, for example, in the safety testing of anti-cancer drugs whose mode of action is to destroy actively dividing cells.(figure 13).
2. For production of proteins : As described above in the examples, the tissue-equivalent
of the present invention has highly enhanced expression of vascular endothelial growth factor
(VEGF) and interleukin-8 (IL-8), a single tissue equivalent secreting about 40 ng VEGF and
450-1000 ng IL-8 in 24 hours. Thus, the tissue equivalent is a good in vitro factory for
producing these proteins.
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Thus, in the foregoing description, specific embodiments of the present invention have been disclosed. It is apparent that various modifications and substitutions could be made to the present invention, which would not be departures from the central concept of the present invention, that of temporarily filling or blocking the pores of a porous matrix with a substance that can be maintained in a solid or semi-solid state, which would not allow cells to pass through, before seeding cells, for the purpose of forming a cellular sheet on one side of the matrix and preventing cell loss through the pores, and also, later removal of the blocking substance from the pores of the porous matrix. Some such modifications or substitutions would be the use of different blocking substances, the use of method other than temperature variation for blocking and unblocking, the use of different temperatures than those presented, the use of different concentrations of blocking substance, the use of different experimental system or design, the use of different method for blocking and unblocking pores based on the properties of the blocking substance. Likewise, sheet formation over a support should be possible at other higher densities than mentioned here. A person with skill in the art can easily devise adaptations of the present method, based on the above central theme. Therefore, although only the described embodiments have been brought forth, they serve the purpose of example or illustration only, and should not be construed as limiting the present invention.
Dated this g day of DaLi/nlWr 2006
For Reliance Life Sciences Pvt. Ltd.
K.V. Subramaniam President
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CLAIMS:
1. A process for preparing a three dimensional tissue equivalent comprising a cellular sheet of dermal fibroblasts cultured adhered over a porous scaffold, wherein the cellular sheet is a high-density macromass cultured sheet, having in vivo and in vitro use.
2. A process as per claim 1, wherein the cellular sheet is entirely on one side of the porous scaffold.
3. A process as per claim 1, wherein the dermal fibroblasts are seeded at a high cell density in the range of 1 x 10 cells per cm to 12 x 10 cells per cm to form the cellular sheet.
4. A process as per claim 1, wherein the porous scaffold comprises a sponge selected from but not limited to gelatin, chitosan, collagen, polyglycolic acid, polylactic acid or alginate.
5. A process as per claim 4, wherein the porous scaffold is preferably chitosan.
6. A process as per claim 1, wherein the cellular sheet is adherent and non contractile.
7. A process as per claim 1, wherein the pores of the porous scaffold in the final tissue-
equivalent are 'open' or devoid of integrated matter occupying them.
8. A process as per claim 1, wherein the tissue equivalent has enhanced expression VEGF and IL-8 as compared to the monolayers.
9. A process as per claim 1, wherein at least 98% dermal fibroblasts do not express HLA-DR surface protein.
10. A process as per claiml, wherein the dermal fibroblasts are at least 95% viable upto 72
hours at 2-8°C.

A process as per claiml, wherein the tissue equivalent does not require cryopreservation.
A process as per claim 1, wherein there is direct contact of the cellular sheet with the wound bed.
A process as per claim 1, wherein the porous scaffold does not impede the diffusion of growth factors from the cells to the wound.
A process as per claim 1, wherein the porous scaffold can hold moisture and enables gas exchange.
A cell friendly process for the preparation of a three dimensional tissue equivalent comprising a cellular sheet of dermal fibroblasts on a porous scaffold comprising the steps:
a. Temporary absorption of a blocking agent into the porous scaffold to fill the
pores,
b. Seeding of the cells on the surface of the porous scaffold, at a density 1 x 106 cells
per cm to 12 x 10 cells per era, to form a macromass cellular sheet while
keeping the blocking agent solidified; and
c. Removal of the blocking agent to result in a cellular sheet entirely on one side of
the porous scaffold;
characterized in that there is no cell loss during seeding of the cells on the porous scaffold,
A process as per claim 15, wherein the blocking agent is selected from gelatin, alginate, pectin, agar and agarose.
A process as per claim 15, wherein the blocking agent preferably is gelatin.

18. A process as per claim 15, wherein the porous scaffold is selected from gelatin,
chitosan, collagen, polyglycolic acid, polylactic acid, alginate.
19. A process as per claim 18, wherein the porous scaffold preferably is chitosan.
20. A process as per claim 15, wherein the temporary absorption of the blocking agent into the porous scaffold comprises steps of
a. pouring of the molten solution of the blocking agent on the porous scaffold,
b. allowing the blocking agent to enter and fill the pores of the porous scaffold; and
c. solidification of the blocking agent.
21. A process as per claim 15, wherein the formation of the cellular sheet over the porous
scaffold comprises the steps of
a. Seeding of the cells at a high density of 1 x 10 cells per cm to 12 x 10 cells per
cm2 on the surface of the porous scaffold the pores of which have been blocked
with solidified blocking agent.
b. Formation of the cellular macromass tissue-like sheet.
c. Removal of the blocking agent.
22. A process for three-dimensional tissue equivalent comprising a cellular sheet of dermal
fibroblasts over a porous sponge as claimed above exemplified herein substantially in the
examples and figures.
Dated this o day of (MumLir- 2006 For Reliance Life Sciences Pvt. Ltd.
K.V. Subfamaniam President

ABSTRACT
The present invention provides a three-dimensional tissue equivalent for in-vivo and in-vitro uses. The three dimensional tissue equivalent of the present invention is a non-contractile cellular sheet cultured over a porous scaffold by a specially designed process wherein the cell sheet is entirely on one side of the porous sponge. In particular, the present invention provides a dermal wound dressing which comprises high cell density.

Documents:

2013-MUM-2006-ABSTRACT(GRANTED)-(7-3-2013).pdf

2013-mum-2006-abstract-1.jpg

2013-mum-2006-abstract.doc

2013-mum-2006-abstract.pdf

2013-MUM-2006-CLAIMS(AMENDED)-(12-7-2010).pdf

2013-MUM-2006-CLAIMS(AMENDED)-(27-12-2010).pdf

2013-MUM-2006-CLAIMS(AMENDED)-(9-1-2013).pdf

2013-MUM-2006-CLAIMS(GRANTED)-(7-3-2013).pdf

2013-MUM-2006-CLAIMS(MARKED COPY)-(9-1-2013).pdf

2013-mum-2006-claims.doc

2013-mum-2006-claims.pdf

2013-mum-2006-correspondance-received.pdf

2013-mum-2006-correspondence(22-1-2009).pdf

2013-MUM-2006-CORRESPONDENCE(28-12-2010).pdf

2013-mum-2006-correspondence(ipo)-(29-12-2009).pdf

2013-MUM-2006-CORRESPONDENCE(IPO)-(8-3-2013).pdf

2013-mum-2006-description (complete).pdf

2013-MUM-2006-DESCRIPTION(GRANTED)-(7-3-2013).pdf

2013-mum-2006-drawing(17-9-2008).pdf

2013-MUM-2006-DRAWING(GRANTED)-(7-3-2013).pdf

2013-mum-2006-drawing.pdf

2013-mum-2006-form 13(27-12-2010).pdf

2013-MUM-2006-FORM 13(9-1-2013).pdf

2013-mum-2006-form 18(17-9-2008).pdf

2013-MUM-2006-FORM 2(GRANTED)-(7-3-2013).pdf

2013-MUM-2006-FORM 2(TITLE PAGE)-(12-7-2010).pdf

2013-MUM-2006-FORM 2(TITLE PAGE)-(GRANTED)-(7-3-2013).pdf

2013-MUM-2006-FORM 3(22-1-2009).pdf

2013-mum-2006-form-1.pdf

2013-mum-2006-form-2.doc

2013-mum-2006-form-2.pdf

2013-mum-2006-form-3.pdf

2013-mum-2006-form-5.pdf

2013-MUM-2006-MARKED COPY(12-7-2010).pdf

2013-MUM-2006-MARKED COPY(27-12-2010).pdf

2013-MUM-2006-OTHER DOCUMENT(27-12-2010).pdf

2013-MUM-2006-REPLY TO EXAMINATION REPORT(12-7-2010).pdf

2013-MUM-2006-REPLY TO EXAMINATION REPORT(27-12-2010).pdf

2013-MUM-2006-REPLY TO HEARING(9-1-2013).pdf

2013-MUM-2006-SPECIFICATION(AMENDED)-(12-7-2010).pdf


Patent Number 255598
Indian Patent Application Number 2013/MUM/2006
PG Journal Number 10/2013
Publication Date 08-Mar-2013
Grant Date 07-Mar-2013
Date of Filing 08-Dec-2006
Name of Patentee RELIANCE LIFE SCIENCES PRIVATE LIMITED
Applicant Address DHIRUBHAI AMBANI LIFE SCIENCES CENTRE, R-282, TTC AREA OF MIDC, THANE BELAPUR ROAD, RABALE, NAVI MUMBAI
Inventors:
# Inventor's Name Inventor's Address
1 MANISHA SHARADCHANDRA DESHPANDE RELIANCE LIFE SCIENCES PVT. LTD. DALC, PLOT NO R-282 TTC AREA OF MIDC, RABALE, NAVI MUMBAI - 400 701,
2 SITHAMRAJU HARINARAYANA RAO Reliance Life Sciences Pvt. Ltd. DALC, Plot No R-282 TTC Area of MIDC, Rabale, Navi Mumbai - 400 701 Maharashtra, India
3 PRALHAD BALASAHED WANGIKAR Reliance Life Sciences Pvt. Ltd. DALC, Plot No R-282 TTC Area of MIDC, Rabale, Navi Mumbai - 400 701 Maharashtra, India
4 PUSHPA VIKRAM KUCHROO Reliance Life Sciences Pvt. Ltd. DALC, Plot No R-282 TTC Area of MIDC, Rabale, Navi Mumbai - 400 701 Maharashtra, India
PCT International Classification Number C12N5/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA