A biotherapeutic GEL for restoring the damaged tissues

Abstract

The present invention provides a biotherapeutic gel that is used for treating chronic, non-healing wounds The present invention is related to a biotherapeutic gel, comprising a combination of immune cells, a biomaterial, a synthetic polymer, and anti-microbial agents. The present invention also provides a process of preparing the biotherapeutic gel from immune cells having wound healing properties.

Description

[0001] The said application is improvement and modification of earlier filed patent application no 202121006270 dated 15 February 2021.

[0002] A BIOTHERAPEUTIC GEL FOR RESTORING THE DAMAGED TISSUES

[0003] FIELD OF THE INVENTION

[0004] The present invention is in the field of a therapeutic cell gel generated from immune cells and the method of treatment accelerating wound healing for normal and chronic wounds. More particularly, the present invention provides a biotherapeutic gel, comprising of immune cells, a biomaterial, synthetic polymers, and anti-microbial agents. The biotherapeutic gel based immunotherapeutic product of the present invention shows properties of restoring and healing damaged tissues, such as wounds.

[0005] BACKGROUND OF THE INVENTION

[0006] Wound healing is a physiologic, very complex phenomenon that occurs when skin integrity is lost and consequently also the barrier function of skin is impaired. This may occur quite often since skin is much exposed to external factors, and the need to avoid systemic infections drives a rapid defence mechanism. Through physiologic healing, the normal status of the skin can be fully recovered, although only a maximum of 70% of previous tensile strength is usually achieved.

[0007] A chronic wound is a wound that does not heal in an orderly set of stages and in a predictable amount of time, or wounds that do not heal within three months are often considered chronic. Chronic wounds often occur in patients with diabetes mellitus due to the impairment of wound healing. This generally has negative consequences for both the patient and the medical system. Growing prevalence of diabetes in society has become a main concern, and it has become a significant medical, social, and economic burden. Impaired healing in diabetes is the result of complex pathophysiology involving vascular, neuropathological, immune, and biochemical components. Hyperglycemia correlates with stiffer blood vessels, which cause slower circulation and microvascular dysfunction, causing reduced tissue oxygenation. Such blood vessel alterations observed in diabetic patients account for reduced leukocyte migration into the wound, which becomes more vulnerable to infections. The hyperglycaemic environment compromises leucocyte function.

[0008] Diabetes mellitus is a metabolic disorder that causes high blood sugar and impedes normal steps of wound healing. It may be caused by either inadequate insulin in the bloodstream, classifying it as Type 1 diabetes mellitus, or by the inability of the insulin molecule to act effectively on target tissue, classifying it as Type 2 diabetes mellitus, which can be due to conformational changes in the structure of insulin or the presence of defective insulin receptors. Currently, there are more than 422 million people worldwide suffering from diabetes, particularly in low-and middle-income countries, and 1.6 million deaths are directly attributed to diabetes each year. The increase in diabetes mellitus patients implies an increase in diabetic complications. Foot ulcers are a major common complication of diabetes mellitus.

[0009] Diabetic foot is one of the most significant and devastating complications of diabetes, which is characterized by ulceration of the lower limb with neuropathy and / or peripheral arterial disease. Diabetic patients have a 15-25% lifetime risk of developing diabetic foot ulcers, of which 40-80% become so severely infected it involves the bone, leading to osteomyelitis. This condition is more prevalent in diabetic patients due to impaired wound healing and persistent inflammatory responses. The condition is more frequent in older patients. It is also the major cause of diabetes associated amputation, which required prolonged hospitalization and management. Early recognition and treatment of foot ulcers in diabetic patients is very critical for the successful outcome of the treatment.

[0010] Conventional treatments for diabetic foot ulcers (DFU) include debridement of the wound, management of any infection, revascularization procedures when indicated, and off-loading of the ulcer. Other methods have also been used as add-on therapies, such as hyperbaric oxygen therapy, use of advanced wound care products (skin grafts, growth factors, matrix proteins), and negative-pressure wound therapy. These treatments and therapies have not been effective enough to reduce the amputation rates, which urges the need for additional treatment. Recently, light has been shed on stem cell therapy as a new technique for treating diabetic lower limb ischemic disease, and specifically, diabetic foot ulcer. Stem cell therapy aims to support wound healing by stimulating the formation of new blood vessels that increase blood supply and relieve limb ischemia. Its effect is through promoting, angiogenesis, secreting paracrine factors, stimulating vascular differentiation, suppressing inflammation, improving collagen deposition, and immunomodulation. Immune cell-based therapies are the most promising approaches to cure certain types of chronic diseases. Immune cells like dendritic cells and T cells have been widely used in clinics, and they have been approved by the FDA for therapeutic purposes.

[0011] Novel biological therapies for the treatment of diabetic ulcers can be classified into the following groups: growth factors, stem cells, anti-diabetic drugs, biological tissues, enzymes, plant products, and immunological molecules. Biological therapy and the use of human skin equivalents seem like promising treatments for chronic ulcers. Such therapies add additional cells and growth factors to a deficient environment of wound healing. Thus, there is a continuous need for developing therapies for treatment of the chronic wound.

[0012] Topical formulations are pharmaceutical formulations that can be applied directly to the skin. Topical formulations are made up in a vehicle, or base, which may be optimized for a particular site of the body or type of skin condition. The product may be designed to be moisturizing or to maximize the penetration of an active ingredient, often a medicine, into or through the skin. There are various topical formulations that can be developed, some of which are creams and lotions, gels, ointments, transdermal patches, sprays, and powders.

[0013] The approach to treating diabetic foot ulcers must be logical as well as systematic. Biological therapy and the use of human skin equivalent (HSE) seem like promising treatments for chronic ulcers. Such therapies add additional cells and growth factors to a deficient environment of wound healing. The biological therapy shows promising treatment potential, as long as it doesn’t block the formation of new vascular tissues. Also, fibroblasts possess the capacity to produce extracellular matrix, which is limited in human skin equivalent. Different combinations of therapies in particular sequences must be used after a complete assessment of the wound in concern, in order to obtain optimum healing.

[0014] Thus, there is a continuous need for developing a biotherapeutic formulation that can give a better result in the treatment of the diabetic foot ulcer. The inventors of the present invention have found a biotherapeutic gel composition for the treatment of foot ulcers.

[0015] OBJECTIVE OF THE INVENTION

[0016] Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:

[0017] The main objective of the present invention is to provide a biotherapeutic gel generated from lymphoid and myeloid cells in vitro.

[0018] Another objective of the present invention is to provide a process of preparing the biotherapeutic gel generated from lymphoid and myeloid cells in vitro.

[0019] Another objective of the present invention is to provide a biotherapeutic gel comprising of immune cells, a biomaterial, synthetic polymers, and anti-microbial agents.

[0020] Another objective of the present invention is to provide a biotherapeutic gel that is simple and cost-effective.

[0021] Another objective of the present invention is to provide a biotherapeutic gel with better patient compliance and improved therapeutic efficacy. Another objective of the present invention is to provide a biotherapeutic gel for restoring and healing damaged tissues.

[0022] SUMMARY OF THE INVENTION

[0023] The present invention provides cells, compositions, and a method of administering the composition to an affected subject a therapeutically effective number of immune cells or the gel composition to achieve accelerated wound healing.

[0024] The main aspect of the present invention is to provide a biotherapeutic gel generated from lymphoid and myeloid cells in vitro.

[0025] Another main aspect of the present invention is to provide a biotherapeutic gel, comprising of immune cells, a bio material, synthetic polymers, and antimicrobial agents.

[0026] Another aspect of the present invention is to provide a biotherapeutic gel composition comprising immune cells, cell culture medium, saline, plasma or serum, antimicrobial agents, biomaterials, and synthetic polymers.

[0027] Another main aspect of the present invention is to provide a biotherapeutic gel and the process for preparing the same.

[0028] Another aspect of the present invention is to provide a biotherapeutic gel for the treatment of chronic wound healing, such as damaged myocardium, damaged brain tissue, damaged spinal cord tissue, and damaged corneal tissue.

[0029] BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1: Shows analysis of the cellular composition of the isolated blood cells using flow cytometry after staining with different fluorescent molecule labelled antibodies. PB2332, PB2333, PB2334 are the three different individual’s blood cells used in the study. The control row indicates the isotype stained cells and the antibody row indicates specific fluorescent antibodies. The percentage of different cell population can be seen in the quadrant or gated analysis.

[0031] Figure 2: Shows analysis of the cellular composition of the collected cultured adherent blood cells using flow cytometry after staining with different fluorescent molecule labelled antibodies. PB 2332, PB2333, PB2334 are the three different individual’s blood cells used in the study. The control row indicates the isotype- stained cells and the antibody row indicates specific fluorescent antibodies. The percentage of different cell population can be seen in the quadrant or gated analysis.

[0032] Figure 3: Shows analysis of the cellular composition of the collected cultured total blood cells using flow cytometry after staining with different fluorescent molecule labelled antibodies. PB2332, PB 2333, PB2334 are the three different individual’s blood cells used in the study. The control row indicates the isotype stained cells and the antibody row indicates specific fluorescent antibodies. The percentage of different cell population can be seen in the quadrant or gated analysis.

[0033] Figure 4: Shows analysis of the cellular composition of the collected adherent or total cells cultured in the absence of growth factor (GMCSF) using flow cytometry after staining with different fluorescent molecule labelled antibodies. The percentage of different cell population can be seen in the gated analysis.

[0034] Figure 5: Shows analysis of cellular composition of the collected adherent or total cells cultured in the presence of growth factor (GMCSF) using flow cytometry after staining with different fluorescent molecule labelled antibodies. The percentage of different cell population can be seen in the gated analysis.

[0035] Figure 6: Shows the appearance of cells before mixing of the gel.

[0036] Figure 7: Image of the prefilled syringe with the biotherapeutic cell gel. Figure 8: Representative images showing the healing of wound observed on different days following the gel treatment.

[0037] Figure 9: Image of the wound of a patient who had chronic non-healing wound at the lower limb. A. Prior to the treatment, B. Four weeks after the treatment

[0038] DESCRIPTION OF THE INVENTION

[0039] Embodiments, of the present disclosure, will now be described with reference to the accompanying drawing.

[0040] Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details, are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0041] The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and / or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.

[0042] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed elements.

[0043] The present invention relates to a biotherapeutic gel generated from immune cells in vitro, process of preparing the biotherapeutic cell gel and the method of treating chronic wounds with the same.

[0044] As used herein, the term "formulation" or “composition” unless otherwise defined refers to gel for topical pharmaceutical dosage forms of the invention.

[0045] As per one embodiment, the term “biotherapeutic” used herein is defined as a term involving the treatment of diseases by means of substances secreted by or derived from living organisms.

[0046] As per one embodiment, the term “biocompatible” used herein is a term describing the property of a material being compatible with living tissue. Biocompatible materials do not produce a toxic or immunological response when exposed to the body or bodily fluids.

[0047] As per one embodiment, the term “plasma or serum” used herein is a plasma with high concentration of platelets, which contains a large amount of proteins which enhance body’s natural healing response. Serum is the liquid that remains after the blood has clotted and consists of 90% water with dissolved hormones, proteins, minerals, and carbon dioxide.

[0048] As per one embodiment, the term “cell culture medium” used herein is a growth medium used in cell culture which support the growth of microorganisms, cells, or small plants and having an appropriate source of energy and compounds which regulate the cell cycle.

[0049] As per one embodiment, the term “T-cells” used herein are immune cells developed from lymphomyeloid niches and lymphocyte harbouring macrophages of in vitro cultures and stem cells in the bone marrow that protect the body and fight against infections in the body, secrete cytokines, and help other immune cells to perform their immune related functions.

[0050] As per one embodiment, the term “macrophages” used herein are lymphocyte harboring macrophages, macrophages of lymphomyeloid niches, and conventional macrophages, are a type of white blood cell and immune cell that kills microorganisms, removes dead cells, secretes growth factors and cytokines, and stimulates the action of cells of immune system and different tissues and organs in the body. They play important role in immunity, immunomodulation, and regeneration of damaged tissues.

[0051] As per one another embodiment, the term “monocytes and monocytoid cells” used herein are a type of white blood cells that reside in the blood and tissues to detect and destroy germs (viruses, bacteria, fungi and protozoa), eliminate infected cells, and differentiate to become macrophages.

[0052] As per one another embodiment, the term “autologous source” used herein is the source of cells or tissues from the patient’s own immune cells.

[0053] As per one another embodiment, the term “allogenic source” used herein is the source of cells, tissue or organs derived from antigenically dissimilar individuals from the same species.

[0054] The main embodiment of the present invention is to provide a biotherapeutic gel for restoring and healing the damaged tissue. As per the main embodiment of the present invention, the biotherapeutic gel comprises of immune cells, a biomaterial, synthetic polymers, and anti-microbial agents.

[0055] As per another embodiment of the present invention, the said immune cells is mainly a mixture of macrophages, monocytes, and T-cells.

[0056] As per another embodiment of the present invention, the said macrophages are lymphocyte harboring macrophages, macrophages of lymphomyeloid niches, and conventional macrophages.

[0057] As per another embodiment of the present invention, the said T-cells are from lymphomyeloid niches and lymphocyte harbouring macrophages of in vitro cultures and stem cells in the bone marrow.

[0058] As per another embodiment of the present invention, the said monocytes and monocytoid cells are white blood cells that reside in the blood and tissues.

[0059] As per another embodiment of the present invention, the said immune cells used are from autologous as well as allogenic sources.

[0060] As per another embodiment of the present invention, the said immune cells used are in the range of O.lxlO6to lOxlO6, more preferably O.lxlO6to 5xl06and most preferably 0.5xl06to 4xl06immune cells.

[0061] As per another embodiment of the present invention, the said biomaterial is biocompatible, wherein the said biomaterial is compatible with the body tissues and cells. As per another embodiment of the present invention, the said biocompatible biomaterial is collagen.

[0062] As per another embodiment of the present invention, the said collagen used is in the range of 1 to 30 mg, more preferably in the range of 5 to 20 mg, and most preferably in the range of 10 to 15 mg.

[0063] As per another embodiment of the present invention, the said synthetic polymer is selected from alginic acid, carbomer (carbopol), CMC, gelatin, methylcellulose, plastibase, and poloxamer, more preferably Carbopol 934.

[0064] As per another embodiment of the present invention, the said synthetic polymer used is in the range of 1 to 20 mg, more preferably in the range of 1 to 15 mg, and most preferably in the range of 5 to 10 mg.

[0065] As per another embodiment of the present invention, the said gel composition comprises a combination of antibiotics. The said combination of antibiotics helps to prevent contamination of the dressing with mold, fungi, or bacteria.

[0066] As per another embodiment of the present invention, the said antimicrobial agents are antibiotics used as a preservative of the gel.

[0067] As per another embodiment of the present invention, the said anti-microbial agents used for preparing the biotherapeutic gel are selected from the group consisting of gentamicin, kanamycin, penicillin, streptomycin, doxycycline, tetracycline, ciprofloxacin, amoxicillin, cefuroxime, and cefepime, or a combination thereof.

[0068] As per another embodiment of the present invention, the said antimicrobial agents are combinations of the antibiotics penicillin, streptomycin, and gentamicin. As per one another embodiment of the present invention, the cells are stimulated with granulocyte-macrophage colony stimulating factor (GMCSF) to increase the yield of macrophage type cells. Flow cytometry is performed to analyze the yield of the stimulated and non- stimulated cells. Figures 1 to 5 show the comparison results of stimulated and non- stimulated cells.

[0069] As per one embodiment of the present invention, the cell culture medium used for growing the cells are selected from MEM, DMEM, F12, DMEM / F12, IMDM, M- 199, RPMI medium, serum free medium, T cell medium, stem cell medium, keratinocyte culture medium, cell specific culture medium, normal saline, ringer lactate solution, phosphate buffered solution, balanced salt solution, and combinations thereof.

[0070] As per one embodiment of the present invention, the plasma or serum used for preparing the cell based biotherapeutic products may be selected from the human or animal origin, autologous or allogeneic, purified serum or plasma components. In the present invention, autologous plasma is the most preferred plasma.

[0071] Another main embodiment of the present invention provides a method of preparing the biotherapeutic products of the present invention in the form of gel.

[0072] As per another embodiment of the present invention, the process for preparing the biotherapeutic gel comprises the following steps:

[0073] 1) Process for preparing the cells: a) Collecting blood from a patient or donor in a blood tube with anticoagulant; b) Bringing the collected blood of step (a) in cool ice packs to the lab within 48 hours; c) Mixing the blood tubes of step (b) gently by inverting 2-3 times; d) Centrifuging the blood tubes of step (c) at a relative centrifugal force of 400g for 15 minutes; e) After centrifugation, separate the plasma above the buffy coat and save the plasma in a new tube; f) Centrifuging the plasma of step (e) at a relative centrifugal force of 500g for 15 minutes and storing at minus 20 degrees Centigrade; g) Diluting the buffy coat and red blood cell layer obtained in step (e) using an equal volume of Dulbecco phosphate buffered saline (DPBS) and mixing it well; h) Subjecting the mixture of step (g) to ficoll gradient centrifugation for separating the mononuclear cells and washing it to obtain purified cells; i) Mixing the final cell pellet obtained in step (h) with 5 mL of growth medium and counting the cells to determine the number of cells; j) Diluting the cells of step (i) in the culture medium with serum at the concentration of 3-4 million per ml and seeding it in the culture flasks, and addition of growth factor GMCSF was an optional step; k) Incubating the flask of step (j) in the incubator with 5% CO2 at 37°C for 5-7 days and then harvesting the cells; l) Harvesting both adherent and non-adherent cells of step (k) from the flask in a sterile centrifuge tube and pelleting it by centrifugation; m) Washing the cell pellet of step (1) using DPBS and characterizing them for the viability, count, and markers. ) Process for preparing the gel base: a) Mixing 0.117 grams of carbopol polymer in 1.5 mL of sterile water and mixing them with a sterile spatula and vortexing it; b) Adding 0.21 gm weight of sterile collagen powder to 4 mL of sterile water and 258 pL of N / 10 HCL solution, vortexing the mixture, and sonicating the same; c) Mixing the steps (a) and (b) and adding the same volume of RPMI 1640 culture media (1: 1) and mixing it; d) Adding drop wise IM NaOH in the mixture of step (c) and adjusting the pH to 7.0; e) Adding 2.5 mL of IX DPBS and 1.2 mL of 5X DPBS in step (d), followed by mixing and vortexing by maintaining the pH around 7.0 with 1 M NaOH; f) Mixing the gel of step (f) with RPMI 1640 culture medium containing plasma; g) Centrifuge the gel of step (f) by centrifuging the tube at 800g for 20 minutes; h) Discarding the supernatant of step (g); i) Storing the gel of step (h) in the refrigerator at 2-8°C.

[0074] 3) Process for preparing the biotherapeutic gel: a) Mixing the cells of step (m) and gel of step (h), for making the final biotherapeutic gel with immune cells; b) Mixing uniformly the gel containing cells of step (a) with 200 units penicillin, 200 micrograms streptomycin, and 60 micrograms gentamicin to get the final product; c) Packing the biotherapeutic gel of step (b) in a suitable container.

[0075] As per another embodiment of the present invention, the biotherapeutic gel comprises lxl06to 10xl06immune cells, 1 to 30 mg collagen, 1 to 30 mg of carbopol 934, 1 to 500 units penicillin, 1 to 500 microgram streptomycin, 1 to 150 microgram gentamicin, 0.01 to 0.1 ml of hydrochloric acid, 0.01 to 0.1 ml of sodium hydroxide, 0.05 ml to 0.2 ml 5 X Dulbecco phosphate buffered saline, 0.4 ml to 0.8 ml RPMI medium, and 0.05 to 0.04 ml water in 1 ml of gel.

[0076] As per another embodiment of the present invention, the biotherapeutic gel can be stored in a suitable container, which can be selected from but is not limited to a prefilled syringe, tube, containers, and bottles.

[0077] As per one embodiment of the present invention, the final gel containing cells can be loaded in a syringe, which is to be closed with a leur-lock cap, after that the prefilled syringe can be stored at 2-8°C in a cold condition. As per another embodiment of the present invention, filling the biotherapeutic gel in a sterile tube or a syringe under sterile aseptic conditions and storing it at 2-8°C until use in patients. The product should be used within 48 hours from the dispatch from the manufacturing laboratory. Using the product for local topical application over the wound following cleaning and debridement and applying the dressing to retain the gel on the wound.

[0078] As per one embodiment, the biotherapeutic gel of the present invention is used for the immunotherapeutic treatment of chronic non-healing wounds, which are selected from the group consisting of damaged tissues such as wounds, non-healing ulcers, damaged myocardium, damaged brain tissue, damaged spinal cord tissue, and damaged corneal tissue, diabetic foot ulcers, burns, an infected tissue or wounds, vascular ulcers, arterial ulcers, infarction, necrosis, gangrene, and bed sore.

[0079] As per one another embodiment, the said biotherapeutic gel of the present invention is used in complete or partial form, wherein the said complete form of gel comprises both cells as well as the gel and the partial form comprises only gel or only cells.

[0080] As per one another embodiment, the biotherapeutic gel of the present invention is applied to the wound, comprising the following steps: a. Cleaning the wound with an antiseptic antimicrobial solution; b. Cleaning the wound with saline solution; c. Applying biotherapeutic cell gel over the cleaned wound of step (b) and spreading the gel over the entire wound; d. Applying transparent dressing on the gel covered wound of step (c); e. Applying a gauze pad or cotton pad over the dressing of step (d) and completely covering the wound; f. Applying the gauze and rolling around the gauze or cotton pad of step (e) to secure the entire dressing; g. Keeping the dressing on the wound for 5 to 10 days if there is no infection; h. Removing the dressing and cleaning the wound with sterile saline; i. Covering the wound with sterile gauze wetted with sterile saline; j. Applying the gauze pad, cotton pad or transparent dressing over the wet gauze and applying the bandage; k. Repeating steps h, i and j once every 3 days until the wound closes completely; l. If there is any sign of infection, such as foul smelling exudates and discoloration of the wound, the wound may need to be cleaned with an antimicrobial solution, and administration of systemic antibiotics may be needed.

[0081] The invention is further illustrated by the following examples which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

[0082] EXAMPLES

[0083] EXAMPLE 1: PREPARATION OF CELL BASED COMPOSITION

[0084] For generating the cellular components of the proposed biotherapeutic gel product, the preparation comprises the following steps: a) The blood was collected from a patient or a donor in blood collection tubes or a blood bag with anticoagulant; b) The collected blood of step (a) brought in cool ice packs to the lab within 24 hours; c) The blood tubes of step (b) were mixed gently by inverting 2-3 times; d) The tube of step (c) was centrifuged at a relative centrifugal force of 400g for 15 min. e) After centrifugation, the plasma above the buffy coat was separated and saved in a new tube; f) The plasma tube of step (e) was centrifuged at a relative centrifugal force of 500g for 15 min and stored at minus 20 degrees Celsius; g) The buffy coat and red blood cell layer obtained in step (e) were diluted using an equal volume of Dulbecco phosphate buffered saline (DPBS) and mixed well; h) The mixture of step (g) was subjected to standard ficoll gradient centrifugation for separating the mononuclear cells; i) The final cell pellet of step (h) was mixed in 5 mL of growth medium and counted the cells to determine the number of cells; j) The cells of step (i) were diluted in the culture medium with serum at concentration of 3-4 million per ml and seeded in the culture flasks. The addition of growth factors such as GMCSF was an optional step; k) The flasks of step (j) were incubated in the incubator with 5% CO2 at 37°C for 5-7 days and then harvested the cells; l) Both adherent and non-adherent cells of step (k) were harvested from the flask in a sterile centrifuge tube and pelleted by centrifugation; m) The cell pellet of step (1) was washed using DPBS and characterized for viability, count, and markers.

[0085] EXAMPLE 2: PREPARATION OF GEL BASED COMPOSITION

[0086] For preparing the gel, parts 1 and 2 were made separately and then mixed. a) For making part 1 , 0.117 g of carbopol polymer was added to 1.5 mL of sterile water, and mixed with a sterile spatula, followed by vortexing it; b) For making part 2, 0.21 g of sterile collagen powder was added to 4 mL of sterile water and 258 pL of N / 10 HCL solution, vortexed the mixture, and sonicated the same; c) Part 1 of step (a) and part 2 of step (b) were mixed, and the same volume of RPMI 1640 culture media (1: 1) was added and mixed; d) IM NaOH was added dropwise in step (c) and adjusted to pH 7.0; e) 2.5 mL of IX DPBS and 1.2 mL of 5X DPBS were added in step (d) and mixed and vortexed by maintaining the pH around 7.0 with 1 M NaOH; f) The gel of step (f) was mixed with RPMI 1640 culture medium containing plasma; g) The gel of step (f) was concentrated by centrifuging the tube at 800g for 20 minutes; h) The supernatant of step (g) was discarded; i) The gel of step (h) was stored in refrigerated condition at 2-8°C.

[0087] EXAMPLE 3: PREPARATION OF BIOTHERAPEUTIC GEL

[0088] FORMULATION a) The cells from step (m) and gel from step (h) were added and mixed for making the final biotherapeutic gel with immune cells; b) The gel containing cells of step (a) was mixed uniformly with 200 units penicillin, 200 micrograms streptomycin, and 60 micrograms gentamicin to get the final product; c) The biotherapeutic gel of step (b) was packed in a suitable container.

[0089] EXAMPLE 4: OPTIMIZATION OF THE BIOTHERAPEUTIC GEL COMPOSITION

[0090] Optimization of the biotherapeutic gel formulation was done using different components and processes to identify the most suitable composition to be delivered in the wound. Different experiments were done to ensure the best cell viability and lowest toxicity.

[0091] A. Effect of only biomaterials on the cells

[0092] Experiment -1:

[0093] For the first experiment, we took a 50 ml tube and added 20 ml of Milli Q water. To the water, we added 1 gram of collagen powder and another 20 ml of Milli Q water. We mixed this solution very well and vortexed for 7-8 times. After mixing, we centrifuged this mixture at 2500 rpm for 5 min. At the end of centrifugation, we got 10 ml of gel. We discarded the supernatant and increased the volume up to 45 ml with Milli Q water. After mixing the solution very well, we centrifuged it at 2500 rpm for 5 min. At the end of centrifugation, we got 10 ml gel again. We discarded the supernatant and added 30 ml of RPMI media. We mixed well and stored it at 4°C for 2 hours. After 2 hours, we centrifuged the tube at 2500 rpm for 10 min. At the end, we got 4 ml of gel. The next day, the gel was sonicated for 7 minutes and examined for cell toxicity after 24 hours.

[0094] Experiment -2:

[0095] Further for the second experiment, 2 grams of collagen powder and 35 ml of Milli Q water were added in a 50 ml tube. Mixed the mixture well and vortexed for 7-8 times. After mixing, the mixture was centrifuged at 2500 rpm for 10 min and got 18 ml of gel. After discarding the supernatant, Milli Q water was added to make the volume up to 45 ml. We centrifuged the mix at 2500 rpm for 15 min and got 22 ml of gel. Again, we discarded the supernatant and increased the volume up to 45 ml with RPMI media. After storing at 4°C for 2 hours, we centrifuged the mix at 2500 rpm for 10 min. We got 8 ml of gel and discarded the supernatant. Finally, we sonicated the gel for 17 min and it was examined for cell toxicity after 24 hours.

[0096] Experiment -3:

[0097] Further for the third experiment, 25 ml of Milli Q water and 1 gm of collagen powder were added in a 50 ml tube. We added H2O to make the volume up to 40 ml and vortexed the same. We incubated the mixture for 5 min at room temperature before centrifugation at 3000 RPM for 15 min. We discarded the supernatant and added Milli Q water up to 40 ml before incubating for 10 min. at room temperature. We got 11 ml of gel after centrifugation at 3000 RPM for 15 min. We discarded the supernatant and added 10 ml RPMI media in each tube before incubating for 5 min. at room temperature. We centrifuged it at 3000 rpm for 15 min and got 5 ml of gel. After washing with RPMI media, the amount of gel was continuously decreasing, and the gel seemed to be toxic to the cells. We saw all cells were dead when examined in PI dye under fluorescent light.

[0098] Experiment -4:

[0099] We took a 15 ml centrifuge tube and added 4 ml of warm Milli Q water. The volume was made up to 13 ml after adding 1 gm collagen powder. After mixing well, the mixture was centrifuged at 2500 rpm for 10 min. We discarded the supernatant and got 10 ml of gel. The gel was divided equally (5 ml) into two tubes, and water was added in each tube to get the volume up to 13 ml. The mixture was centrifuged at 2500 rpm for 5 min. After centrifugation we got a gel volume of 3 ml in each tube. We added 670 microliters of 10 X RPMI media and mixed it. Then we added 30 microliters of IM NaOH for setting the pH to 7. After that, we took the tubes out of the biosafety cabinet and did sonication for 7.5 min. at 37 °C.

[0100] Result:

[0101] For the cytotoxicity study, we took 2 ml of cell solution from the culture flask and pelleted it by centrifugation. We added 0.5 ml of culture media for suspending the cells before adding 2 microliters of red dye (DIL). We incubated the mixture in a CO2 incubator for 20 min. After 20 min., add 8 ml of DPBS for washing and centrifuge at 2000 rpm for 10 min. We discarded the supernatant and added 150 microliters of culture media and 450 microliters of gel prepared previously. We stored the mixture at 4°C for 12 hours. When we examined the gel after washing with medium, the amount of gel had decreased. We saw in fluorescent light after 12 hours; we found all cells were dead. The biomaterial chitosan was also tested for its suitability in the formulation at 1 and 2 percent concentration. The results showed that it has cytotoxicity on the cells. Thus, it was omitted from the formulation.

[0102] B. Effect of only polymer on the cells

[0103] To study the effect of different polymers, we took a 50 ml centrifuge tube and added 0.117 g of Carbopol 974. After adding 5.5 ml of water to the tube, we mixed with a spatula. We vortexed the mixture for approx. 5-10 minutes and it became semi-solid. Equal volume of culture media (1: 1) was added and mixed well. Then we added drop wise 1 molar NaOH for pH setting at 7.0 (approx. 1-1.3 ml 1 molar NaOH had to be added). Total gel volume became 11 ml. To this, 2.5 ml of IX DPBS was added, followed by 1.2 ml of 5X DPBS. All this was mixed well and vortexed. Total gel volume was 14 ml. We added a double volume of RPMI media to the gel and mixed well. We incubated the mixture for 5 minutes and then centrifuged it. After centrifugation, we discarded the supernatant. The gel was very thin, and we used gel for cytotoxicity checks without washing. At 24 hours, we found 85% of cells viable. The experiment was carried out with double the concentration (0.234 gm) of the polymer, and the results showed that the gel was very thin and there was no separation in the gel after centrifugation. Similarly, the gel was prepared using the 0.117 gm and 0.234 gm of carbopol 940. At the lower concentration, the gel was very thin and showed 70% cell viability at 24 hours. At higher concentrations, the gel was very thin, and there was no separation.

[0104] Result: We tested the carbopol 934 in the same concentration as the other polymers and found the lower concentration was optimal and the viability of the cells was above 90 percent, and it was further used in the formulation to combine with the biomaterial for delivering the cells.

[0105] C. Optimization of cell viability

[0106] We took a 15 ml centrifuge tube and 0.117 g of Carbopol 934. After adding 5.5 ml of water to the tube, we mixed it with a spatula. We vortexed the mixture for 5-10 min. It became semi- solid. We took a 15 ml centrifuge tube and added 4 ml of water with 144 pL of 0.1N HCL. We mixed it and added 0.215 g of collagen powder. We mixed for 20 min. and sonicated for 5 minutes at 35°C. Then we mixed the collagen and carbopol for 2 to 3 minutes to remove any lumps. To this we added dropwise IM NaOH to get to a pH of 7.2. We added 2.5 ml of IX DPBS mix and 1.2 ml of 5X DPBS. Everything was mixed. The resultant gel was smooth and thick. We took 3 ml of LB media and 30 microliters of gel which were incubated at 37°C for 7 days, and there was no growth of any bacteria during this period. The result showed the mixture was sterile.

[0107] Now, further to check the cell viability, the following experiments were carried out: Tube 1: We took 2.5 ml of gel, 2.5 ml of RPMI media and added 180 microliters of IM NaOH. We mixed well and got the pH of 7.0. Tube 2: We took 2.5 ml of gel, 2.5 ml of 1 X DPBS, and added 180 microliters of IM NaOH. We mixed well and got the pH of 7.0.

[0108] Tube 3: We took 2.0 ml of gel and added 144 uL of IM NaOH. We mixed well and got the pH of 7.0.

[0109] Tube 4: We took 0.5 ml of gel from Tube 2 and 0.5 ml of media. After mixing, we centrifuged at 8000 rpm for 10 min. After 10 minutes we discarded the supernatant. Tube 5: We took 0.5 ml of Tube 2 gel and added 0.5 ml of IX DPBS solution. After mixing, we centrifuged at 8000 rpm for 10 min. After 10 minutes we discarded the supernatant.

[0110] Tube 6: We took 0.5 ml of Tube 2 gel and added 1 ml of IX DPBS solution. After mixing, the gel was centrifuged. After 10 minutes we discarded the supernatant.

[0111] Tube 7: We took 0.5 ml of Tube 1 gel and added 1 ml of culture media. After mixing, we centrifuged and discarded the supernatant.

[0112] For the cytotoxicity testing of tubes 1-4, we used the cells with a concentration of 22.30xl06 / mL cells. We took 180 microliters of gel and 20 microliters of cells stored at 4°C. In tube 4, viable cells were seen. For the cytotoxicity testing of tubes 5-7, we used cell suspension with a concentration of 7.50xl06 / ml cells. We took 180 microliters of gel and 20 microliters of cells stored at 4°C. It was found that after 18 hours, in tubes 5 and 6, all cells were dead. In tube 7, 82.3% of cells were viable at 42 hours, which was the last observation. The results of the cytotoxicity of the gels are given in the table below.

[0113] Table 1: Cytotoxicity of the gels under different preparation conditions.

[0114] Tube / Time No. of Live cells No. of Dead cells Viability

[0115] Tube: - 1

[0116] 2 hours 0 Cells All Cells are dead 0 %

[0117] Tube: - 2

[0118] 2 hours 0 Cells All Cells are dead 0 %

[0119] Tube: - 3 2 hours 0 Cells All Cells are dead 0 %

[0120] Tube: - 4

[0121] 2 hours 208 Cells 58 Cells 77.3 %

[0122] 19 hours 17 Cells 132 Cells 11.4 %

[0123] Tube: - 5

[0124] 2 hours 0 Cells All Cells are dead 0 %

[0125] Tube: - 6

[0126] 2 hours 0 Cells All Cells are dead 0 %

[0127] Tube-7

[0128] 18 hours 65 Cells 11 Cells 85.5 %

[0129] 21 hours 107 Cells 9 Cells 92.3 %

[0130] 42 hours 72 Cells 18 Cells 82.3 %

[0131] D. Improving the gel suitability

[0132] Further to improve the gel suitability, we added culture media double in the volume of the gel and labeled the gel as 6A. After mixing and incubating the gel for 5 min, we centrifuged and discarded the supernatant. The final gel volume used was 14 ml.

[0133] We used 26.90 X 10 million / ml cells. We took 180 microliters of gel and 20 microliters of cells stored at - 4°C. The viability was 93.3% at 72 hours. Similarly, we added two volumes of the gel and one volume of RPMI media and labeled the gel as 6B. After mixing and centrifuging, the gel was tested for cytotoxicity. We took 200 microliters of gel and 20 microliters of cells stored at 4°C. All cells were dead at

[0134] 48 hours. Further, an equal volume of RPMI media mixed with the gel was labeled as 6C and centrifuged the same. The result of toxicity showed that all cells were dead in 48 hours. In gel 6D, RPMI media with plasma are mixed with an equal volume of the gel. The cytotoxicity study showed that viability was 83.7% at 24 hours, but all cells were dead at 48 hours. In 6E, to RPMI media with one third part of plasma mixed with the gel and centrifuged. The cytotoxicity study showed a cell viability of 86% at 72 hours. Result: We can thus conclude that, the gel labeled as 6A having a double volume of culture media is more viable in comparison to others for 72 hours. The results for the same are given in the below table. Table 2: Cytotoxicity of the gels under different preparation conditions

[0135] Gel No / Time No. of live Cells No. of dead Cells Viability

[0136] Gel 6A

[0137] 5 hours 227 Cells 8 Cells 97.2 %

[0138] 48 hours 130 Cells 10 Cells 92.8 %

[0139] 72 hours 99 Cells 7 Cells 93.3 %

[0140] Gel 6B

[0141] 5 hours 13 Cells 25 Cells 34.2 %

[0142] 48 hours All Cells were Dead

[0143] Gel 6C

[0144] 5 hours 20 Cells 45 Cells 30.7 %

[0145] 48 hours All Cells were Dead

[0146] Gel 6D

[0147] 24 hours 258 Cells 50 Cells 83.7 %

[0148] 48 hours All Cells were Dead

[0149] Gel 6E

[0150] 24 Hours 669 Cells 79 Cells 89.4 %

[0151] 48 Hours 519 Cells 64 Cells 85 %

[0152] 72 Hours 600 Cells 90 Cells 86 %

[0153] EXAMPLE 5: FINAL FORMULATION OF THE BIOTHERAPEUTIC GEL

[0154] The biotherapeutic cell gel of the present invention was prepared by using immune cells from different donors with the mentioned composition and with the mentioned process. The final composition of the biotherapeutic cell gel (each gram) is as given below: Table 3: Final formulation of the biotherapeutic gel

[0155] S. No. Ingredients Quantity

[0156] 1. Immune cells 0.5 x 106

[0157] 2. Collagen 10 mg

[0158] 3. Carbopol 934 5 mg

[0159] 4. Plasma 0.1 ml

[0160] 5. Penicillin 200 units

[0161] 6. Streptomycin 200 microgram

[0162] 7. Gentamicin 60 microgram

[0163] 8. Hydrochloric acid 0.02 ml

[0164] 9. Sodium hydroxide 0.03 ml

[0165] 10. Dulbecco phosphate buffered saline 0.1 ml

[0166] 11. RPMI medium 0.55ml

[0167] 12. Water 0.2 ml

[0168] EXAMPLE 6: STABILITY OF THE BIOTHERAPEUTIC CELL GEL

[0169] The prepared biotherapeutic cell gel was tested for stability by measuring the cell viability at different time intervals, and the results are given in the below table:

[0170] Result:

[0171] Table 4: Results for stability of the biotherapeutic gel

[0172] Gel ID 8 Hours 24 Hours 48 hours 72 Hours 96 Hours

[0173] G001 94 % -

[0174] G002 96 % 96 % 97.9 %

[0175] G003 96 % 95.9%

[0176] G004 - 99 % 96.8 %

[0177] G005 - 99.2 % 98.3 %

[0178] G006 - 96.6 % 95.6 %

[0179] G007 - 99.7 % 99.6 %

[0180] G008 - - 96.7 % G009 - - 98.9 %

[0181] G010 - - 97.7 %

[0182] G011 - 98.1 % 98 % 94.5 %

[0183] G012 - 96.6 % 86.8 % 85.7 %

[0184] G013 - 98.1% 98% 94.5%

[0185] G014 - 96.7% 96.2% 82%

[0186] G015 - 99.4% 96.8% 81.1% 59.2%

[0187] G016 - 99.3% 96.2% 86.5% 60.5%

[0188] G017 - 95.5% 87.3% 67.6 %

[0189] The formulated product was found to be stable for up to 48 hours with more than 90% cell viability and up to 72 hrs with more than 80% cell viability.

[0190] EXAMPLE 7: IN VIVO VALIDATION OF THE SAFTY AND EFFICACY OF BIOTHERAPEUTIC CELL GEL PRODUCT IN CHICKEN MODEL

[0191] A) SAFETY EVALUATION:

[0192] The safety of the cell gel was tested in a preclinical study using chickens as a model. First, the effect of gel without any cells was studied on the intact skin of healthy chickens and then tested on wounds created in the skin of healthy chickens. We created a small 4 square centimeter wound in the inner side of the wing and applied the gel to the wound and observed for any sign of inflammation or adverse reaction for 5 days.

[0193] Results: The results showed no sign of any inflammation or adverse reaction at the site of application. These studies showed that the gel prepared for delivering the cells is safe to use without any adverse reaction.

[0194] B) EFFICACY EVALUATION:

[0195] Further in vivo preclinical validation for understanding the efficacy of biotherapeutic gel products was done in a chicken model. For studying the biotherapeutic efficacy of the gel formulation of the product, 4 to 6-week old broiler chickens were used. Blood was collected, mononuclear cells were isolated, and cultured to generate the required cells. On day 0, a wound was created by removing a piece of 4 square centimeters of skin tissue using a sterile scalpel on both sides of the body under the wings, and one was used for treatment with our therapeutic product and the other was used as a control. The cells were harvested, biotherapeutic gel was prepared and applied to the wound, and dressing was applied. In each bird, the wound created on the left side of the body was applied with 2 million cells containing gel on the wound, whereas the wound on the right side served as control. The dressing was removed on day 4, and the wound was cleaned with normal saline once every 2 days afterwards. The birds were followed up till day 10, and image were taken on day 10. The healing of the wound was also monitored by measuring the wound size on day 0 and comparing it with the size on day 10. The area of wound was measured by measuring the length at the longest position and the width at the shortest position of wound in millimeters (mm) using a scale. The wound area was estimated by multiplying the width and length and expressed in square (Sq) mm. The size of wounds on days 0 and 10 is given in the below table, and a representative image of the wound is given in Figure 8.

[0196] Results:

[0197] Table 5: Results regarding reduction in wound size in chicken model

[0198] S. DAY-0 DAY-10 % of Wound

[0199] No. (Wound area in Sq (Wound area in Sq Reduction mm) mm)

[0200] Treated Untreated Treated Untreated Treated Untreated

[0201] 1. 400 399 10 108 98 73

[0202] 2. 441 400 42 154 90 62

[0203] 3. 399 441 10 144 97 67

[0204] 4. 420 399 35 169 92 58

[0205] Mean 415 410 24 144 94 65 The results showed that the percentage reduction in the treated wound was 94% compared to 73% in the untreated wound. The average wound area in the treated and untreated wounds was 415 and 410 sq. mm on day 0, and it was 24 and 114 sq. mm on day 10, respectively.

[0206] EXAMPLE 8: CLINICAL STUDY OF THE BIOTHERAPEUTIC GEL PRODUCT

[0207] Further, the gel was used as a therapy on two patients who were suffering from chronic non-healing ulcers. The first patient was diabetic and suffering from a nonhealing diabetic foot ulcer, and then underwent a lower limb amputation from the knee joint. The wound at the knee following amputation was not healing for many months, and he was in a wheelchair with limited mobility. The wound was not healing despite trying conventional therapies for many months, and the patient was not able to go forward with the plan to wear a prosthetic limb. The patient’s wound was not responding to standard therapies. Upon the clinician’s guidance, blood was collected, the therapy was prepared and applied to the wound, and dressing was done. After four weeks following single application of the therapy, the wound healed completely. Under the guidance of the clinician, we applied the therapy in another patient who was suffering from a chronic non-healing wound in the lower limb for many months and not responding to standard treatments. If the wound was not healing, he would have to go for amputation of the lower limb. This patient’s wound was also healed completely after four weeks following the single treatment, as shown below in Figure 9.

[0208] Conclusion: The present invention thus provides a biotherapeutic gel for the treatment of chronic wounds. The biotherapeutic gel of the present invention comprising of immune cells, a biomaterial, synthetic polymers and anti-microbial agents. The present invention also provides the process for preparing the biotherapeutic gel.

Claims

CLAIMSI Claim,1. A biotherapeutic gel for restoring the damaged tissues, comprising of immune cells, a biomaterial, synthetic polymers, and anti-microbial agents.

2. The biotherapeutic gel as claimed in claim 1, wherein said immune cells is mainly a mixture of macrophages, monocytes, and T-cells.

3. The biotherapeutic cell gel as claimed in claim 1, wherein said immune cells used are in the range of 0.1xl06to 10 xlO6, more preferably O.lxlO6to 5xl06, most preferably 0.5xl06to 4 xlO6immune cells in 1 ml gel.

4. The biotherapeutic gel as claimed in claim 1, wherein said biomaterial is collagen.

5. The biotherapeutic gel as claimed in claim 1, wherein said collagen is used in the range of 1 to 30 mg, more preferably in the range of 5 to 20 mg, and most preferably in the range of 10 to 15 mg.

6. The biotherapeutic gel as claimed in claim 1, wherein said synthetic polymer is selected from alginic acid, carbomer (carbopol), CMC, gelatine, methylcellulose, plastibase, poloxamer, more preferably carbopol 934.

7. The biotherapeutic gel as claimed in claim 1, wherein said synthetic polymer used is in the range of 1 to 30 mg, more preferably in the range of 1 to 20 mg, and most preferably in the range of 5 to 15 mg.

8. The biotherapeutic gel as claimed in claim 1, wherein said anti-microbial agents used for preparing the cell based therapeutic gel were selected from the group consisting of gentamicin, kanamycin, penicillin, streptomycin, doxycycline, tetracycline, ciprofloxacin, amoxicillin, cefuroxime, and cefepime, or a combination thereof.

9. The biotherapeutic gel as claimed in claim 1, wherein said penicillin used is in the range of 1-500 units, more preferably in the range of 100-400 units, and most preferably in the range of 150-250 units.

10. The biotherapeutic gel as claimed in claim 1, wherein said streptomycin used is in the range of 1-500 micrograms, more preferably in the range of 100-400 micrograms and most preferably in the range of 150-250 micrograms.

11. The biotherapeutic gel as claimed in claim 1, wherein said gentamicin used is in the range of 1-150 micrograms, more preferably in the range of 1-100 micrograms and most preferably in the range of 50-70 micrograms.

12. A process for preparing the biotherapeutic gel, comprises the following steps:1) Process for preparing the cells a) Collecting blood from a patient or donor in a blood tube with anticoagulant; b) Bringing the collected blood of step (a) in cool ice packs to the lab within 24 hours; c) Mixing the blood tubes of step (b) gently by inverting 2-3 times; d) Centrifuging the blood tubes of step (c) at a relative centrifugal force of 400g for 15 minutes; e) After centrifugation, separate the plasma above the buffy coat and save the plasma in a new tube; f) Centrifuging the plasma of step (e) at a relative centrifugal force of 2500 RPM or 500g for 15 minutes and storing at minus 20 degrees Celsius; g) Diluting the buffy coat and red blood cell layer obtained in step (e) an using an equal volume of Dulbecco phosphate buffered saline (DPBS) and mixing it well; h) Subjecting the mixture of step (g) to ficoll gradient centrifugation for separating the mononuclear cells; i) Mixing the final cell pellet obtained in step (h) with 5 mL of growth medium and counting the cells to determine the number of cells; j) Diluting the cells of step (i) in the culture medium with serum at the concentration of 3-4 million per ml and seeding it in the culture flasks, and the addition of growth factors such as GMCSF, is an optional step; k) Incubating the flask of step (j) in the incubator with 5% CO2 at 37°C for 5-7 days and then harvesting the cells; l) Harvesting both adherent and non-adherent cells of step (k) from the flask in a sterile centrifuge tube and pelleting it by centrifugation;m) Washing the cell pellet of step (1) using DPBS and characterizing them for the viability, count, and markers.2) Process for preparing the gel base: a) Mixing 0.117 grams of carbopol polymer in 1.5 mL of sterile water and mixing them with a sterile spatula and vortexing it; b) Adding 0.21 gm weight of sterile collagen powder to 4 mL of sterile water and 258 pL of N / 10 HCL solution, vortexing the mixture, and sonicating the same; c) Mixing the steps (a) and (b) and adding the same volume of RPMI 1640 culture media (1: 1) and mixing it; d) Adding dropwise IM NaOH in the mixture of step (c) and adjusting the pH to 7.0; e) Adding 2.5 mL of IX DPBS and 1.2 mL of 5X DPBS in step (d), followed by mixing and vortexing by maintaining the pH around 7.0 with 1 M NaOH; f) Mixing the gel of step (f) with RPMI 1640 culture medium containing plasma and; g) Concentrating the gel of step (f) by centrifuging the tube at 800g for 20 minutes; h) Discarding the supernatant of step (g); i) Storing the gel of step (h) in the refrigerator at 2-8°C.3) Process for preparing the biotherapeutic gel: a) Mixing the cells of step (m) and gel of step (h), for making the final biotherapeutic gel with immune cells; b) Mixing uniformly the gel containing cells of step (a) with 200 units penicillin, 200 micrograms streptomycin, and 60 micrograms gentamicin to get the final product; c) Packing the biotherapeutic gel of step (b) in a suitable container.

13. The biotherapeutic gel as claimed in claim 1, wherein the said biotherapeutic gel comprises IxlO6to 10 xlO6immune cells, 1 to 30 mg of collagen, 1 to 30 mg of carbopol 934, 1 to 500 units penicillin, 1 to 500 microgramsstreptomycin, 1 to 150 micrograms gentamicin, 0.01 to 0.1 ml of hydrochloric acid, 0.01 to 0.1 ml of sodium hydroxide, 0.05 ml to 0.2 ml 5x Dulbecco phosphate buffered saline, 0.4 ml to 0.8 ml RPMI medium, and 0.05 to 0.4 ml water in 1 ml of gel.

14. The biotherapeutic gel as claimed in claim 1, wherein the said biotherapeutic gel is used for the immunotherapeutic treatment of chronic wounds, such as but not limited to wounds, non-healing ulcers, damaged myocardium, damaged brain tissue, damaged spinal cord tissue, and damaged corneal tissue, diabetic foot ulcers, burns, an infected tissue or wounds, vascular ulcers, arterial ulcers, infarction, necrosis, gangrene, and bed sore.

15. The biotherapeutic gel as claimed in claim 1, wherein the said biotherapeutic gel is applied to the wound, comprises the following steps: a. Cleaning the wound with an antiseptic antimicrobial solution; b. Cleaning the wound with saline solution; c. Applying biotherapeutic gel over the cleaned wound of step (b) and spreading the gel over the entire wound; d. Applying transparent dressing to the gel covered wound of step (c); e. Applying a gauze pad or cotton pad over the dressing of step (d) and completely covering the wound; f. Applying the gauze rolling around the gauze or cotton pad to secure the entire dressing; g. Keeping the dressing on the wound for 5 to 10 days if there is no infection h. Removing the dressing and cleaning the wound with sterile saline; i. Covering the wound with sterile gauze wetted with sterile saline; j. Applying the gauze pad, cotton pad, or transparent dressing over the wet gauze and applying the bandage; k. Repeating steps h, i and j once every 3 days until the wound closes completely; l. If there is any sign of infection, such as foul smelling exudates and discoloration of the wound, the wound may need to be cleaned with anantimicrobial solution, and administration of systemic antibiotics may be needed.

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