WO2011145110A1

WO2011145110A1 - A novel cord blood plasma nutrient formulation and a method for the preparation thereof

Info

Publication number: WO2011145110A1
Application number: PCT/IN2011/000338
Authority: WO
Inventors: Subhadra Dravida; Deepika Akkinepally
Original assignee: Subhadra Dravida; Deepika Akkinepally
Priority date: 2010-05-15
Filing date: 2011-05-16
Publication date: 2011-11-24

Abstract

A novel cord blood plasma nutrient formulations suitable as solvent medium for suspending stem cells for autologous or allogenic transplantation. The plasma may be derived from patient's own umbilical cord blood or from the umbilical cord blood of a related or non-related donor. The cord blood plasma nutrient formulation of the invention is used for therapeutic purposes in treating various degenerative diseases. The invention also relates to a method of preparing the novel formulation comprising collection of umbilical cord blood, extraction of serum / plasma, growth of stem cells, re-suspension of isolated and expanded stem cells for transplantation of the stem cells.

Description

A novel cord blood plasma nutrient formulation and a method for the

preparation thereof

FIELD OF THE INVENTION

The invention relates, to novel cord blood plasma nutrient formulation. More particularly, the invention relates to novel cord blood plasma nutrient formulations suitable as solvent medium for suspending stem cells for autologous or allogenic transplantation. The plasma may be derived from patient's own umbilical cord blood or from the umbilical cord blood of a related or non-related donor. The cord blood plasma nutrient formulation of the invention is used for therapeutic purposes in treating various degenerative diseases.

The invention also relates to a method of preparing the novel formulation comprising collection of umbilical cord blood, extraction of serum / plasma, growth of stem cells, re-suspension of isolated and expanded stem cells for transplantation of the stem cells.

BACKGROUND OF THE INVENTION There are a large number of degenerative diseases, medical conditions or disorders which cannot be cured by existing drug therapies, surgery or transplantation methods.

In recent past years, biotechnology research and development has taken a new dimension. Research scientists and biotechnologists have been continuously working to find out an answer to the non curable diseases in improving the quality of life. One of the fields of biotechnology which has taken a new shape and which is considered to be most relevant development in the field of medicines in recent years is developments and inventions related to stem cells. Stem cells are special cells that have the ability to develop into any type of cells like bone, muscle, nerve, etc. and can be grown into any part of the body as required. Stem cells have the capacity to divide to generate daughter cells that retain the properties of the stem cell or to produce daughter cells that begin to differentiate into a more specialized cell type or to produce one daughter cell of each type. Stem cells are thus central to normal human growth and development and by their intrinsic characteristics these are also potential source of new cells for regeneration of diseased or damaged tissue.

Stem cells are typically found in the embryo and umbilical cord of an organism and in reservoirs within the human body. Stem cells are broadly differentiated into three distinct types i.e. totipotent , pluripotent, and multipotent. At the earliest stages after fertilization (up to the eight cell stage), all cells of the embryo are totipotent i.e., they have the capacity to develop into every type of cell needed for full development of the organism including extra-embryonic tissues such as the placenta and the umbilical cord. Therefore, a fertilized egg is totipotent which produces an entire organism.

The specialized cells obtained after several cycles of cell division of totipotent cells are called pluripotent. With the development of the embryo, pluripotent cells become multipotent. The multipotent cells can specifically produce blood, skin, nerve, or any other types of body cells. While stem cells are extraordinarily important in early human development, multipotent stem cells are also found in children and adults. One example of stem cells present in adults is blood stem cells present in adult's bone marrow. Blood stem cells perform the critical role of continually replenishing the supply of blood cells including red blood cells, white blood cells, and platelets throughout the life span of human being. Adult stem cells have shown very high potential to treat degenerative diseases. Clinical evidence of the potential of stem cells from cord blood origin in treating debilitating diseases suggests that the day is not far when the customized stemcell (SC) therapy products would be available to the person in need. Adult stem cells that are undifferentiated with multipotency are typically isolated from collected biopsy sample and cultured in vitro with established culture conditions with or without plasma. The expanded cell culture is counted for the density and the cells are transplanted at the chosen site for tissue specific grafting. There is no single factor that has shown to be affecting the efficacy of the transplantation outcome in terms of the treatment of the disease targeted with many variations in the clinical trial reports, published.

Safety, feasibility, and efficacy of stem cell (SC) transplantation for a variety of pathological conditions are currently under thorough investigation. There are several studies that use very high numbers of stem cells. Despite the need for vast numbers of human mesenchymal stem cells (MSCs) as therapeutic means, there is only limited information available on presentation of the required transplantation. Presently, there is extensive discrepancy among laboratories concerning the media, the starting and passaging cell-plating density, the culture surfaces, and the addition of supplementary factors for the successful isolation and expansion of SCs that end up with heterogeneous cell populations both in vitro experiments and in clinical trials. In addition to certain critical factors like the methods of isolation, culture conditions, passaging density, fetal calf plasma (FCS) lot selection, population selection, use of basic fibroblast factor (bFGF) for proliferation enhancement, and effect of donor age and cryopreservation, the efficacy of the stem cell culture as the drug for transplantation might also depend on the formulation, which will be administered. The viability of the final stem cell culture for transplantation may depend on the solvent in which the cells are mixed before administering.

By controlling the in vitro culture conditions of the stem cells, the viability along with the retention of the typical characteristic features of the cells can be achieved, till the transplantation, but after grafting, the behavior of the cells at the site may depend on the internal environment along with the intrinsic conditions like the composition of the grafted stem cell produce, survived. Any reagent or solution used for making the clinically transplantable stem cells being biological is acceptable in order to avoid unexplained manifestations in the subjects. Indian Patent No. 220672 discloses a method of culturing mesenchymal stem cells (hMSC) derived from mononuclear cell fractions comprising the steps of plating a mononuclear cell suspension in a culture medium wherein, the culture medium comprises of a mixture of: Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium and; β-FGF and cord blood plasma. Wherein, the Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 are used is in the ratio of 1 : 1.

US Patent 7060494 discloses a method of growing human mesenchymal stem cells (hMSC) comprising the steps of i). separation of umbilical cord and collecting the umbilical cord blood; ii). isolating the plasma from the umbilical cord blood; and, iii). culturing human stem cells in umbilical cord blood plasma. This document discloses that the blood is collected from the umbilical cord at the time of birth, after the infant is separated from the umbilical cord, and the blood is collected from an umbilical vein free of anticoagulants. The blood is collected in a blood bag having a collecting needle which is inserted into the umbilical vein and the blood is allowed to flow from the vein into the blood bag, and the blood is allowed to clot at room temperature and the bag is transported to a processing area which is a cGMP clean room wherein, the blood is allowed to clot up to about eight hours and then centrifuged at 1000 g in a blood bag centrifuge.

WO/2007/141657 discloses a pharmaceutical composition comprising a therapeutic effective amount of human embryonic stem (hES) cells which are free of animal products, feeder cells, growth factors, leukemia inhibitory factor, supplementary mineral combinations, amino acid supplements, vitamin supplements, fibroblast growth factor, membrane associated steel factor, soluble steel factor and conditioned media. The hES and their derivatives are suspended in a pharmaceutically acceptable biocompatible solution.

Prior art documents show that umbilical cord blood has been used for growth and expansion of the stem cells. The broad areas of published patents and papers on Cord blood derived Stem cells (SC), till date, have been on different sources of SC, methods of isolation, culture conditions, conditioning the medium in the expansion of the stem cells to treat specific groups of diseases, devices and methods to collect Cord blood, compositions with the scaffolds and SC, proposed to treat diseases. Although the published papers have discussed the preparation of the clinical grade SC with detailed information on different kinds of media used and different culture conditions, there has never been any disclosure of the formulation of Cord blood derived clinical grade SC for presentation for either autologous or allogenic transplantation.

OBJECT OF THE INVENTION

The primary object of the invention is to provide a novel umbilical cord blood plasma nutrient formulation suitable for use as solvent medium for suspending the stem cells for autologous or allogenic transplantation. Another object of the invention is to provide a novel cord blood plasma nutrient formulation for therapeutic use in regenerative medicine.

A further object of the invention is to provide a method of preparation of cord tissue mesenchymal stem cell and cord blood MNCs culture formulation which is used in treating degenerative diseases.

SUMMARY OF THE INVENTION

A novel cord blood plasma nutrient (CBSN) formulation derived from patient's own umbilical cord blood or from the donated umbilical cord blood plasma. The umbilical cord blood plasma is treated, frozen and thawed up to three times, along with normal saline with re-suspended stem cells (up to 2 millions) for autologous transplantation.

The cord blood plasma nutrient (CBSN) formulation is suitable as solvent medium for suspending the stem cells for autologous or allogenic transplantation. The cord blood plasma nutrient formulation is used for therapeutic purposes in treating various degenerative diseases.

The novel formulation also comprises a derivative of cord blood in addition to the stem cells (SC) derived from the same sample collected.

The cord blood plasma is chemically treated with normal saline and the cord blood stem cells are mixed with normal saline in combination with plasma.

The plasma is prepared from the collected Cord blood from the patient and mixed with sterile normal saline in 1 :1 ratio. 1.0 ml of this nutrient rich solvent is formulated with 1.0 ml of 2 x 10⁶ stem cells for therapy.

The invention also relates to a method of preparing the novel formulation comprising collection of umbilical cord blood, extraction of plasma, growth of stem cells, re- suspension of isolated and expanded stem cells for transplantation of the stem cells.

DETAILED DESCRIPTION

Detailed embodiments of the present invention are disclosed herein however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention.

Certain words and phrases are used repeatedly in the foregoing and forthcoming paragraphs. To bring out clarity about such words and phrases and to avoid any interpretation other than those meant by inventor, by a person skilled in the art, those words and phrases are defined as under.

"Cord blood" means umbilical cord blood.

"Patient" means the person under treatment for life threatening diseases or conditions including but not limited to disease related to blood and immune system, cancer, diabetes, neurodegenerative diseases, pulmonary diseases, cardiovascular, hepatic, cerebrospinal diseases and organ transplant.

"Donated" means when the cord blood nutrient plasma (CBSN) is not derived from the patient's own umbilical cord blood rather, it is derived from the cord blood of some other person who may or may not be related to the patient

The present invention relates to a novel umbilical cord blood plasma formulation - Cord Blood Plasma Nutrient (CBSN) to be used as solvent medium for suspending the stem cells for autologous or allogenic transplantation, wherein the plasma is derived from patient's own or donated umbilical cord blood.

In a preferred embodiment, the plasma (CBSN) is derived from patient's own umbilical cord blood. The patient's own umbilical cord blood plasma is treated, frozen and thawed up to three times, along with normal saline with re-suspended stem cells (up to 2 millions) for autologous transplantation.

In another embodiment of the invention, the formulation comprises donated umbilical cord blood plasma.

The cord blood plasma nutrient is treated mechanically as a nutrient rich solvent medium for re-suspending the isolated and expanded stem cells (SC) for transplantation. It is postulated that the suspension medium used acts as a buffer, positively influencing the viability of the stem cells (SC), in vivo, at the site of injection.

In one embodiment of the invention, the novel formulation comprises a derivative of cord blood in addition to the stem cells (SC) derived from the same sample collected. The cord blood plasma nutrient is chemically treated with normal saline and the cord blood stem cells are mixed with normal saline in combination with plasma nutrient.

The plasma is prepared from the collected Cord blood from the patient and mixed with sterile normal saline in 1 : 1 ratio. 1.0 ml of this nutrient rich solvent is formulated with 1.0 ml of 2 x 10⁶ stem cells for therapy.

The process of preparing the novel umbilical cord blood plasma formulation comprises collecting the cord blood (CB) from the donor following the standard medical procedure including the method for collection of cord blood for plasma separation, harvesting the stem cells by centrifuging the cord blood on ficoll gradient and separating the mononuclear cell (MNC) rich buffy coat for future use and seeding an aliquot of it with tissue culture medium for expansion of mesenchymal stem cells (MSC).

The invention further comprises preserving the separated plasma supernatant from cord blood (CB) by freezing at -70 degree centigrade.

The MNC or MSC harvested at the end of the culture period are washed twice with just saline and pelleted down to count.

The desired number of cells decided as per the weight of the host body is mixed with the thawed CBSN, centrifuged once for sedementing the particles and blood cell debris, if any and normal saline (1 :1). The invention comprises freezing the cord blood (CB) derived plasma nutrient for the purpose of formulation; using the thawed cord blood CBSN for formulating the CB and Cord tissue derived SC for clinical applications and the formulation of the thawed CBSN combined with normal saline along with the clinical grade SCs to make an all biological stem cell product ready for transplantation.

Umbilical Cord Blood Collection

Umbilical cord blood was obtained from healthy term newborns after caesarian delivery at Femi Care Hospital, Hyderabad, A.P, India.

A consent form approved by the Institutional Review Board was signed by the mother whose medical and family history obtained prior to collection did not reveal any exclusion criteria for umbilical cord blood (UCB) donation.

The blood was obtained using gravity bag method wherein, the cord was double clamped, transected, and cleaned with iodine and alcohol.

Blood collection bag containing 49 ml of citrate-phosphate-dextrose-adenine (CPDA) anticoagulant was used to collect the umbilical cord blood (UCB)

A 16-gauge needle from the collection bag was inserted into the umbilical vein; blood was allowed to flow by gravity until the blood flow ceased. The UCB volume was estimated by subtracting the tare weight of the bag and the volume of anticoagulant from the total weight of the blood-containing bag.

Umbilical Cord Tissue (UCT) and Mesenchymal Stem Cell (MSCs) Harvest* Expansion Isolation and Culture of Mesenchymal Stem Cells (MSCs) from Umbilical Cord Tissue (UCT).

The isolation of Mesenchymal Stem Cells (MSCs) was performed as described below:

Example - 1

Method of experiment 1. Umbilical Cord Tissue (UCT) of 1 ^u ₂ inch length is cut under sterile conditions in biosafety cabinet and placed in 60mm x 15mm petri plates.

2. 5ml of Dulbecco's Phosphate Buffer Saline (D-PBS) IX (Gibco^®- 14040) with Penicillin-streptomycin (Gibco^®- 15070) is added to the UCT and incubated at 37° Celsius with 5% C0 for 2 hrs.

3. Then the UCT is transferred to biosafety cabinet and all the clots are removed and cleaned. 4. The UCT is cut open longitudinally and 1ml of 0.05% Trypsin-EDTA (Gibco^®- 25300) is added and incubated at 37° Celsius with 5% C0₂ for 20 minutes.

5. After the incubation time, the UCT is transferred to biosafety cabinet and Dulbecco's Modified Eagle Medium- F 12 - low glucose IX (DMEM - F12) (Gibco^®- 10567) is added to UCT to the enzymatic activity of trypsin.

6. Wharton's Jelly is scooped off from the tissue using a cell scrapper and the liquid containing PBS IX, DMEM and Wharton's Jelly is pipetted off. 7. UCT is washed thrice with PBS IX of 3ml each time in 60 X 15 mm petri plate. 8. The cleaned UCT is then chopped into small chunks of less than 1cm diameter.

9. The orientation of the explants so called chunks are placed in such a way that the mesenchyme stromal surface is in contact with the petri plate and the glossy epithelium is facing upwards.

10. Few drops of medium is added to the explants and incubated at 37° Celsius with 5% C0₂ for 2 hrs.

11. Finally after the incubation period, 1ml of DMEM - F12 medium (combined with 5% Fetal Bovine Plasma (HiClone) and Recombinant Epidermal Growth Factor is added for the Day - 1 procedure.

12. On Day - 3, the cells are observed which start growing from the explants culture. The medium is changed and transferred to CO₂ incubator.

13. As such medium is changed thrice a week for high confluency.

14. The confluency was observed 100% on the 14^th day from the culture. Umbilical Cord Blood Mononuclear Cells (MNCs)

Example - 2

The Cord Blood collecting bag was cleaned superficially and checked for any clots in the bag.

The collection bag was weighed for the amount of cord blood collected and transferred to the bio-safety cabinet.

Then 40 ml of 2 M HAES was added to 100 ml of cord blood collected. The bag was incubated for 10 minutes.

RBC, Buffy Coat and Plasma supernatant were separated using Sepax. The Plasma was freezed at -70° Celsius for the further analysis Buffy Coat bag, rich of MNC's was collected and tube-sealed.

For 22ml of BC bag, 8 ml of DMSO was added slowly by sandwiching the BC bag between the cool gel packs.

Specific BC bag was bar coded and controlled freezing was done and was stored at - 196° C in Liquid Nitrogen Tank.

Cord Blood Plasma & Storage

105 ml of Cord Blood upon separation using Sepax (Biosafe) yielded 55ml of plasma.

The plasma obtained is centrifuged at 3500 rpm for 3-4 minutes to obtain 15 ml of Cord Blood Plasma Nutrient (CBSN) as supernatant.

CBSN is analyzed for the Tabulated parameters below based on Procedural, Temperature Variation.

Example 3

(a) . Collection of the source: The Cord Blood Plasma Nutrient (CBSN) upon centrifugation of Cord Blood Plasma was taken as the source.

(b) . Method of the Experiment: CBSN was thawed at 37° C in water bath for 2-3 minutes. 4mL of the CBSN was transferred into centrifuge tube and kept at - 4 C for the analysis of the below tabulated parameters.

(c). Results:

10 Bilirubin 0.14 rng/ dL 0.2 - 0.8 mg/dL (In direct)

11 Blood Urea 1.86 mg/dL NA

12 Cortisol 0.7 Ug/dL 172 +/- 72 Ug/L

13 Vitamin Bi₂ <45 pg/mL 260 - 310 pg/Ml

14 Vitamin D < 1.2 ng/ mL 25 - 44 nmol/mL

15 Sodium 192 mmol/L 126- 166 mmol/L

16 Potassium 2.1 mmol/L 5.6- 12 mmol/L

17 Chloride 69 mmol/L 96 - 104 mmol/L

18 Bicarbonate 3.00 mmol/L 22.3+/- 2.5 mmol/L

19 Ig G 288 mg/dL 1571+/- 298 mg/dL

20 Ig M 23 mg/dL 10+/- 5 mg/dL

21 Ig A 21 mg/dL NA

Example-4 (a). Collection of the source: The Cord Blood Plasma Nutrient (CBSN) upon centrifugation of Cord Blood Plasma was taken as the source. (b). Method of the Experiment: CBSN was thawed at 37 Celsius in the water bath for 2-3 minutes.

4mL of the Cord blood Plasma supernatant was transferred into centrifuge tube and frozen at - 70 ⁰ Celsius for three hours.

Later the sample was thawed at 37 ⁰ Celsius for 3 minutes and frozen at -70° Celsius for three hours. The process was repeated twice and analyzed for the below parameters.

Result:

Creatinine 0.4 mg/dL

NA

Bilirubin (Total) 0.20 mg/dL

Upto 1.8 mg/dL

Bilirubin (Direct) 0.06 mg/dL

0.0 - 0.2 mg/dL

Bilirubin (In direct) 0.14 mg/ dL

0.2 - 0.8 mg/dL

Blood Urea 2.30 mg/dL NA

Cortisol 0.7 Ug/dL 172 +/- 72 Ug/L

Vitamin B^ <45 pg/mL 260 - 310 pg/Ml

Vitamin D < 1.2 ng/ mL 25 - 44 nmol/mL

Sodium 96 mmol/L 126- 166 mmol/L

Potassium 1.1 mmol/L 5.6- 12 mmol/L

Chloride 66 mmol/L 96 - 104 mmol/L

Bicarbonate 3.00 mmol/L 22.3+/- 2.5 mmol/L

Ig G 288 mg/dL 1571+/- 298 mg/dL

Ig M 23 mg/dL 10+/- 5 mg/dL

Ig A 21 mg/dL NA Example- 5 a). Collection of the source: The Cord Blood Plasma Nutrient (CBSN) obtained upon centrifugation of Cord Blood Plasma was taken as the source.

(b). Method of the Experiment: CBSN was thawed at 37° Celsius.

4mL of the CBSN was transferred into centrifuge tube and kept at -20° C for one hour. Later the sample was thawed at 37 ⁰ Celsius for 3 minutes and frozen at -70° C for three hours. The process was repeated twice and 2ml of each was distributed in two different tubes 3(a) and 3(b).

The 3(a) tube was reconstituted to 4ml with Distilled water at Room Temperature. The 3(b) tube was reconstituted to 4ml with Phosphate Buffer Saline (PBS) at Room Temperature. These two samples were analyzed and following results were obtained.

Sample 3(a)

S. No. Parameters for

Analysis Observed Values Reference Values

1 Total Protein 0.6 g/dL 4.6 +/ - 0.5 g/dL

2 Albumin 0.0 g/dL >3.5 g/dL

3 Total Cholesterol 6 mg/dL <100 mg/dL

4 Blood Sugar 573 mg/dL 70-180 mg/dL

(random) T₄ (Total Thyroxine) 16.98 nmol/L 140.9 nmol/L

Alkaline Phosphatase OU/L 46 +/-12UL

Creatinine 0.0 mg/dL NA

Bilirubin (Total) 0.20 mg/dL Upto 1.8 mg/dL

Bilirubin (Direct) 0.03 mg/dL 0.0 - 0.2 mg/dL

Bilirubin (In direct) 0.17 mg/dL 0.2 - 0.8 mg/dL

Blood Urea 0.46 mg/dL NA

Cortisol 0.3 Ug/dL 172+/- 72 Ug/L

Vitamin B_]2 <45 pg/mL 260-310pg/Ml

Vitamin D < 1.2 ng/ mL 25 - 44 nmol/mL

Sodium 135 mmol/L 126- 166 mmol/L

Potassium 5.0 mmol/L 5.6-12 mmol/L

Chloride 102 mmol/L 96-104 mmol/L

Bicarbonate 3.00 mmol/L 22.3+/- mmol/L

IgG 288 mg/dL 1571+/- 298 mg/dL 20 IgM 23 mg/dL 10+/- 5 mg/dL

21 IgA 21 mg/dL NA

Sample 3 (b)

10 Bilirubin (In direct) 0.08 mg/ dL 0.2 - 0.8 mg/dL

11 Blood Urea 0.46 rng/dL NA

12 Cortisol 0.3 Ug/dL 172 +/- 72 Ug/L

13 Vitamin B₁₂ <45 pg/mL 260 - 310 pg/Ml

14 Vitamin D < 1.2 ng/ mL 25 - 44 nmol/mL

15 Sodium 135 mmol/L 126- 166 mmol/L

16 Potassium 5.0 mmol/L 5.6- 12 mmol/L

17 Chloride 102 mmol/L 96 - 104 mmol/L

18 Bicarbonate 3.00 mmol/L 22.3+/- mmol/L

19 Ig G 288 mg/dL 1571+/- 298 mg/dL

20 Ig M 23 mg/dL 10+/- 5 mg/dL

21 Ig A 21 mg/dL NA

Discussion: This protocol interprets the invention of novel umbilical cord blood plasma nutrient formulation - CBSN as useful solvent medium for suspending the stem cells for both autologus and allogenic transplantation, where in the plasma is of either the own patient or donor. The cord blood plasma nutrient proves to be potential medium to be used as solvent medium for SC suspension.

From the attempt made to analyze the different parameters during procedural Temperature variation the cord blood plasma nutrient with various proteins, vitamins processed can be used as solvent medium to suspend the SC grafting.

Claims

I Claim

1. A novel cord blood plasma nutrient (CBSN) formulation for suspending stem cells for autologous and / or allogenic transplantation and therapeutic use in treatment of degenerative diseases wherein, said formulation is derived from umbilical cord blood.

2. A formulation as claimed in claim 1, wherein said formulation comprises one or more components selected from the group consisting of total protein between 0.1 g/dL to 1.1 g/dL, Albumin about 0.3g/dL, total cholesterol between 6 mg/dL to 7 mg/dL, blood sugar between 573mg/dL to 1072mg/dL, total thyroxin (T₄) between 16.98 nmol/L to 33.98 nmol/L, alkaline phosphatase upto 1 U/L, creatinine upto0.4mg/dL, bilirubin (total) 0.20 mg/dL, bilirubin (direct) between 0.03mg/dL to 0.06 mg/dL, bilirubin (in direct) between 0.14 mg/dL to 0.17 mg/dL, blood urea between 0.46 mg/dL to 2.30 mg/dL, Cortisol between 0.3 Ug/dL to 0.7 Ug/dL, vitamin-Bi₂ < 45pg/ml, vitamin-D < 1.2 ng/mL, sodium between 96mmol/L to 192 mmol/L potassium between 1.1 mmol/L to 5 mmol/L, chloride between 66 mmol/L to 102 mmol/L, bicarbonate about 3mmol/L, Ig G about 288 mg/dL, Ig M about 23mg/dL and Ig A about 21 mg/dL.

3. A formulation as claimed in claim 1, wherein said umbilical cord blood is obtained from the umbilical cord blood of same subject or from a donor.

4. A formulation as claimed in claim 1, wherein said formulation comprises nutrient rich solvent medium for re-suspending isolated and expanded stem cells for both autologous and allogenic transplantation and is obtained by mechanical treatment of the umbilical cord blood.

5. A formulation as claimed in any of the above claims wherein, said umbilical cord blood plasma is chemically treated with normal saline and the cord blood stem cells are mixed with normal saline in combination with plasma.

6. A formulation as claimed in claim 1, wherein said formulation is obtained from cord blood collected from the subject and mixed with normal saline in 1 : 1 ratio.

7. A formulation as claimed in claim 1, wherein said formulation is preserved in freezed conditions at -70° C to -196° C.

8. A formulation as claimed in claim 1, wherein said formulation comprises upto 2 million (2 x 10⁶) stem cells / ml for treatment of disease related to blood and immune system, cancer, diabetes, neurodegenerative diseases, pulmonary diseases, cardiovascular, hepatic, cerebrospinal diseases and organ transplant.

9. A method of preparing the novel formulation as claimed in claim 1, wherein said method comprises collecting the cord blood from the donor; harvesting the stem cells by centrifuging the cord blood on ficoll gradient; separating the mononuclear cell (MNC) rich buffy; and harvesting the mesenchymal stem cells from Cord tissue.

10. A method as claimed in claim 9, wherein the method further comprises freezing the cord blood derived plasma for formulation; using thawed cord blood plasma nutrient for formulating the cord blood derived stem cells for clinical applications; and combining the thawed cord blood derived plasma nutrient with normal saline alongwith the clinical grade stem cells to make all biological stem cell product ready for transplantation.