ENHANCING THE FUNCTION IMMUNOCYTES AND HEMOCYTES USING TGP AND MICROGRAVITY

Abstract

Method of enhancing the cellular functions characterized by the inclusion of at least the process, wherein the aqueous solution containing the cells in the low temperature sol state, in an aqueous solution that exhibits thermoreversible sol-gel transition of being a sol at low temperatures and gel at high temperatures containing at least a hydrogel-forming polymer, bringing the solution containing the to a high temperature gel and then culturing the cells under microgravity.

Claims

1. A method of enhancing functions of cells by embedding them in an aqueous solution containing a novel thermo reversible gelation polymer which at low temperature is in liquid state and at higher temperature is in gel state and by subjecting and culturing the cells under such embedded conditions to microgravity.

Description

EXAMPLES

[0079] Herein below, the present invention will be described in more detail with reference to Examples. However, it should be noted that the present invention is defined by Claims, but is not limited by the following Examples.

Production Example 1

[0080] 10 g of a polypropylene oxide-polyethylene oxide copolymer (average polymerization degree of propylene oxide/ethylene oxide=about 60, Pluronic F-127, mfd. by Asahi Denka K.K.) was dissolved in 30 ml of dry chloroform, and in the co-presence of phosphorus pentaoxide, 0.13 g of hexamethylene diisocyanate was added thereto, and the resultant mixture was subjected to reaction under refluxing at the boiling point for six hours. The solvent was distilled off under reduced pressure, the resultant residue was dissolved in distilled water, and subjected to ultrafiltration by using an ultrafiltration membrane having a molecular cutoff of 3?10.sup.4 (Amicon PM-30) so as to fractionate the product into a low-molecular weight polymer fraction and a high-molecular weight polymer fraction. The resultant aqueous solution was frozen, to thereby obtain a high-polymerization degree product of F-127 and a low-polymerization degree product of F-127.

[0081] When the above high-polymerization degree product of F-127 (TGP-1, a hydrogel-forming polymer according to the present invention) was dissolved in distilled water under ice-cooling in an amount of 8 mass %. When the resultant aqueous solution was gradually warmed, it was found that the viscosity was gradually increased from 21? C., and was solidified at about 27? C. so as to be converted into a hydrogel state. When the resultant hydrogel was cooled, it was returned to an aqueous solution at 21? C. Such a conversion was reversibly and repetitively observed. On the other hand, a solution which had been obtained by dissolving the above low-polymerization degree product of F-127 in distilled water under ice-cooling in an amount of 8 mass %, was not converted into a gel state at all even when it was heated to 60? C. or higher.

Production Example 2

[0082] 160 mol of ethylene oxide was subjected to an addition reaction with 1 mol of trimethylol propane by cationic polymerization, to thereby obtain polyethylene oxide triol having an average molecular weight of about 7000.

[0083] 100 g of the thus obtained polyethyleneoxide triol was dissolved in 1000 ml of distilled water, and then 12 g of potassium permanganate was slowly added thereto at room temperature, and the resultant mixture was subjected to an oxidization reaction at this temperature for about one hour. The resultant solid content was removed by filtration, and the product was subjected to extraction with chloroform, and the solvent (chloroform) was distilled off, to thereby obtain 90 g of a polyethylene oxide tricarboxyl derivative.

[0084] 10 g of the thus obtained polyethylene oxide tricarboxyl derivative, and 10 g of polypropylene oxide diamino derivative (average propylene oxide polymerization degree: about 65, trade name: Jeffamine D-4000, mfd. by Jefferson Chemical Co., U.S.A., cloud point: about 9? C.) were dissolved in 1000 ml of carbon tetrachloride, and then 1.2 g of dicyclohexyl carbodiimide was added thereto, and the resultant mixture was allowed to cause a reaction for 6 hours under refluxing at boiling point. The resultant reaction mixture was cooled and the solid content was removed by filtration, and thereafter the solvent (carbon tetrachloride) therein was distilled off under reduced pressure. Then, the resultant residue was dried under vacuum, to thereby obtain a polymer for coating (TGP-2) comprising plural polypropylene oxide blocks, and polyethylene oxide block combined therewith. This polymer was dissolved in distilled water under cooling with ice so as to provide a concentration of 5 mass %. When the sol-gel transition temperature of the resultant aqueous solution was measured, it was found that the sol-gel transition temperature was about 16? C.

Production Example 3

[0085] 96 g of N-isopropyl acrylamide (mfd. by Eastman Kodak Co.), 17 g of N-aclyloxy succinimide (mfd. by Kokusan Kagaku K.K.), and 7 g of n-butyl methacrylate (mfd. by Kanto Kagaku K.K.) were dissolved in 4000 ml of chloroform. After the purging with nitrogen gas, 1.5 g of N,N-azobisisobutyronitrile was added thereto, and the resultant mixture was subjected to polymerization at 60? C. for 6 hours. The reaction mixture was concentrated, and then was reprecipitated in diethyl ether. The resultant solid content was recovered by filtration, and then was dried under vacuum, to thereby obtain 78 g of poly (N-isopropyl acrylamide-co-N-aclyloxy succinimide-co-n-butyl methacrylate).

[0086] Then, an excess of isopropylamine was added to the thus obtained poly(N-isopropyl acrylamide-co-N-aclyloxy succinimide-co-n-butyl methacrylate) to thereby obtain poly(N-isopropyl acrylamide-co-n-butyl methacrylate). The thus obtained poly(N-isopropyl acrylamide-co-n-butyl methacrylate) had a sol-gel transition temperature of about 19? C. in its aqueous solution.

[0087] Then, 10 g of the thus obtained poly(N-isopropyl acrylamide-co-N-aclyloxy succinimide-co-n-butyl methacrylate) and 5 g of both terminal-aminated polyethylene oxide (molecular weight=6000, mfd. by Kawaken Fine Chemical K.K.) were dissolved in 1000 ml of chloroform, and the resultant mixture was allowed to cause a reaction at 50? C. for 3 hours. The reaction mixture was cooled to room temperature, and thereafter 1 g of isopropylamine was added thereto, and was left standing for 1 hour. The reaction mixture was concentrated, and then was precipitated in diethyl ether. The solid content was recovered by filtration, and thereafter was dried under vacuum, to thereby obtain a polymer for coating (TGP-3) comprising plural poly(N-isopropyl acrylamide-co-n-butyl methacrylate) blocks and polyethylene oxide block combined therewith.

[0088] This polymer was dissolved in distilled water under cooling with ice so as to provide a concentration of 5 mass %. When the sol-gel transition temperature of the resultant aqueous solution was measured, it was found that the sol-gel transition temperature was about 21? C.

Production Example 4

(Sterilization Method)

[0089] 2.0 g of the above-mentioned polymer (TGP-1) was placed in an EOG (ethylene oxide gas) sterilizing bag (trade name: Hybrid Sterilization bag, mfd. by Hogi Medical Co.), and was filled up with EOG by use of an EOG sterilizing device (trade name: Easy Pack, mfd. Inouchi Seieido Co.) and then the bag was left standing at room temperature for twenty-four hours. Further, the bag was left standing at 40? C. for half a day, EOG was removed from the bag and the bag was subjected to aeration. The bag was placed in a vacuum drying chamber (40? C.) and was left standing for half a day, and was sterilized while the bag was sometimes subjected to aeration.

[0090] Separately, it was confirmed that the sol-gel transition temperature of the polymer was not changed even after this sterilization operation.

Production Example 5

[0091] 37 g of N-isopropylacrylamide, 3 g of n-butyl methacrylate, and 28 g of polyethylene oxide monoacrylate (having a molecular weight of 4,000, PME-4000 mfd. by Nihon Yushi K.K. (NOF Corporation)) were dissolved in 340 mL of benzene. Thereafter, 0.8 g of 2,2-azobisisobutyronitrile was added to the resultant solution, and then was subjected to a reaction at 60? C. for 6 hours. 600 mL of chloroform was added to the thus obtained reaction product so as to be dissolved therein, and the resultant solution was dropped into 20 L (liter) of ether so as to be precipitated therein. The resultant precipitate was recovered by filtration, and the precipitate was then subjected to vacuum drying at about 40? C. for 24 hours. Thereafter, the resultant product was again dissolved in 6 L of distilled water. The solution was concentrated to a volume of 2 L at 10? C. by using a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 10?10.sup.4 (HIP100-43 mfd. by Amicon).

[0092] The concentrated solution was diluted with 4 L of distilled water, and then, the dilution operation was carried out again. The above dilution and concentration by ultrafiltration were further repeated 5 times, so as to eliminate products having a molecular weight of 10?10.sup.4 or lower. The product which had not been filtrated by this ultrafiltration (i.e., the product remaining in the inside of the ultrafiltration membrane) was recovered and freeze-dried, so as to obtain 60 g of a hydrogel-forming polymer (TGP-4) according to the present invention having a molecular weight of 10?10.sup.4 or higher.

[0093] 1 g of the thus obtained hydrogel-forming polymer (TGP-4) according to the present invention was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of the obtained aqueous solution was measured, it was found to be 25? C.

Production Example 6

[0094] The hydrogel-forming polymer (TGP-3) according to the present invention which had been obtained in Production Example 3 was dissolved so as to provide a concentration of 10 mass % in distilled water. When the steady flow viscosity ? thereof at 37? C. was measured, it was found to be 5.8?10.sup.5 Pa.Math.sec. In the measurement of the steady flow viscosity q, a stress rheometer (AR-500), and an acryl disk (diameter: 4 cm) as a measuring device were used. The thickness of a sample was set to 600 ?m, and applying a shearing stress of 10 N/m.sup.2, the resultant creep was measured for 5 minutes after 5 minutes had passed.

[0095] On the other hand, agar was dissolved so as to provide a concentration of 2 mass % in distilled water at 90? C., and the mixed solution was converted into a gel state at 10? C. for 1 hour. Thereafter, ? thereof at 37? C. was measured. As a result, the obtained value exceeded the measurement limit (1?10.sup.7 Pa.Math.sec) of the apparatus.

Production Example 7

[0096] 71.0 g of N-isopropylacrylamide and 4.4 g of n-butyl methacrylate were dissolved in 1,117 g of ethanol. To the resultant mixture solution, an aqueous solution which had been obtained by dissolving 22.6 g of polyethylene glycol dimethacrylate (PDE 6000, mfd. by NOF Corporation) in 773 g of water was added. The oresultant solution was heated to 70? C. under a nitrogen stream. While the solution was maintaining at 70? C. under a nitrogen stream, 0.8 mL of N,N,N,N-tetramethylethylenediamine (TEMED) and 8 mL of 10% ammonium persulfate (APS) aqueous solution were added to the solution, and then was subjected to a reaction for 30 minutes under stirring. Further, 0.8 mL of TEMED and 8 mL of 10% APS aqueous solution were added thereto 4 times at 30-minute intervals, and the polymerization reaction was terminated. The reaction mixture was cooled to 10? C. or lower, it was diluted with 5 L of cold distilled water with a temperature of 10? C. Thereafter, the solution was concentrated to 2 L at 10? C., by using an ultrafiltration membrane with a molecular weight cutoff of 10?10.sup.4.

[0097] 4 L of cold distilled water was added to the concentrated solution for dilution, and the above concentration operation using the ultrafiltration was conducted again. Thereafter, the above dilution and ultrafiltration concentration were repeated 5 times, so as to eliminate products with a molecular weight of 10?10.sup.4 or lower. The product which had not been filtrated by the above ultrafiltration (product remaining in the ultrafiltration membrane) was recovered and freeze-dried, so as to obtain 72 g of the hydrogel-forming polymer (TGP-5) according to the present invention with a molecular weight of 10?10.sup.4 or higher.

[0098] 1 g of the thus obtained hydrogel-forming polymer (TGP-5) according to the present invention was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, it was found to be 20? C.

Production Example 8

[0099] 42.0 g of N-isopropylacrylamide and 4.0 g of n-butyl methacrylate were dissolved in 592 g of ethanol. To the resultant mixture solution, an aqueous solution which had been obtained by dissolving 11.5 g of polyethylene glycol dimethacrylate (PDE 6000, mfd. by NOF Corporation) in 65.1 g of water was added. The resultant solution was heated to 70? C. under a nitrogen stream. While the solution was maintained at 70? C. under a nitrogen stream, 0.4 mL of N,N,N,N-tetramethylethylenediamine (TEMED) and 4 mL of 10% ammonium persulfate (APS) aqueous solution were added to the solution, and then, the thus obtained solution was subjected to a reaction for 30 minutes under stirring. Further, 0.4 mL of TEMED and 4 mL of 10% APS aqueous solution were added thereto 4 times at 30-minute intervals, and the polymerization reaction was terminated. The reaction mixture was cooled to 5? C. or lower, it was diluted with 5 L of cold distilled water with a temperature of 5? C. Thereafter, the solution was concentrated to 2 L at 5? C., by using an ultrafiltration membrane with a molecular weight cutoff of 10?10.sup.4.

[0100] 4 L of cold distilled water was added to the concentrated solution for dilution, and the above concentration operation using the ultrafiltration was conducted again. Thereafter, the above dilution and ultrafiltration concentration were repeated 5 times, so as to eliminate The product with a molecular weight of 10?10.sup.4 or lower. The product which had not been filtrated by the above ultrafiltration (product remaining in the ultrafiltration membrane) was recovered and freeze-dried, so as to obtain 40 g of the hydrogel-forming polymer (TGP-6) according to the present invention with a molecular weight of 10?10 or higher.

[0101] 1 g of the thus obtained hydrogel-forming polymer (TGP-6) according to the present invention was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, it was found to be 7? C.

Production Example 9

[0102] 45.5 g of N-isopropylacrylamide and 0.56 g of n-butyl methacrylate were dissolved in 592 g of ethanol. To the resultant mixture solution, an aqueous solution which had been obtained by dissolving 11.5 g of polyethylene glycol dimethacrylate (PDE 6000, mfd. by NOF Corporation) in 65.1 g of water was added. The resultant solution was heated to 70? C. under a nitrogen stream. While the solution was maintained at 70? C. under a nitrogen stream, 0.4 mL of N,N,N,N-tetramethylethylenediamine (TEMED) and 4 mL of 10% ammonium persulfate (APS) aqueous solution were added to the solution, and then was subjected to a reaction for 30 minutes under stirring. Further, 0.4 mL of TEMED and 4 mL of 10% APS aqueous solution were added thereto 4 times at 30-minute intervals, and the polymerization reaction was terminated. The reaction mixture was cooled to 10? C. or lower, it was diluted with 5 L of cold distilled water with a temperature of 10? C. Thereafter, the solution was concentrated to 2 L at 10? C., by using an ultrafiltration membrane with a molecular weight cutoff of 10?10.sup.4.

[0103] 4 L of cold distilled water was added to the concentrated solution for dilution, and the above concentration operation using the ultrafiltration was conducted again. Thereafter, the above dilution and ultrafiltration concentration were repeated 5 times, so as to eliminate The product with a molecular weight of 10?10.sup.4 or lower. The product which had not been filtrated by the above ultrafiltration (product remaining in the ultrafiltration membrane) was recovered and freeze-dried, so as to obtain 22 g of the hydrogel-forming polymer (TGP-7) according to the present invention with a molecular weight of 10?10.sup.4 or higher.

[0104] 1 g of the thus obtained hydrogel-forming polymer (TGP-7) according to the present invention was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, it was found to be 37? C.

Example 1

[0105] NK cells cultured in normal medium under normal gravity (a) and microgravity (b) for 6 hours and NK cells cultured in TGP medium of sol-gel transition temperature of 20? C. for 6 hours under normal gravity (c) and microgravity (d) were compared for cell population and NO level, and FACS analysis results.

[0106] Peripheral blood was collected from autoimmune enhancement therapy (AIET) patients as per the national regulatory guidelines, after informing them and with their consent. Separation of cells and culturing was carried out in a GMP conformant class 10,000 clean room. The collected peripheral blood was centrifuged at 3,000 rpm for 10 min at room temperature, the supernatant plasma and the precipitated hemocyte pellets were separately transferred to sterile centrifuge tubes. The plasma was inactivated by heat treatment at 56? C. for 30 minutes, and then centrifuged at 3,000 rpm for 10 minutes at room temperature. The supernatant plasma was placed in a sterile centrifuge tube and stored at ?20? C. To separate the mononuclear cells, phosphate buffer (PBS) was mixed well with the hemocyte pellets that were placed in the sterile centrifuge tube, in a capacitance ratio of 1:1. In addition, a lymphoprep solution (Axis-Shield, Norway, Cat # LYS 3773) was overlaid on the hematocyte PBS mixture (i.e., 15 mL of lymphoprep solution and 30 mL of hematocyte PBS mixture) in a capacitance ratio of 1:2. The centrifuge tube was centrifuged in room temperature at 1,500 rpm for 10 min. The peripheral blood mononuclear cells (PBMNCs) were pipetted out and transferred to a sterile container so as not to disturb the separation layer. The PBS was added to the PBMNCs in a capacitance ratio of 2:1. After centrifuging at 1,200 rpm for 10 minutes in room temperature, the supernatant was discarded. The precipitated pellet was gently mixed with 30 mL of PBS. 20 ILL of PBS dispersion of the PBMNCs was taken out and the cell population was measured. The remainder was centrifuged at 1,200 rpm for 10 minutes in room temperature, the supernatant was discarded and the PBS was added and stored for cell culturing. The cell population was measured using trypan blue exclusion method. The PBMNCs was dispersed in a predetermined volume of Optimizer media ((Invitrogen, USA)) so as to achieve a cell density of 1?10?6 cells/mL, cultured at 39? C. for 24 hours in a NK cell culturing flask (Biotherapy Institute, Tokyo) and thereafter at 37? C. for 24 hours under 5% CO2. The cells in the flask were centrifuged at room temperature of 1,200 rpm for 8 min along with the flask washing PBS solution. The NK cells were dispersed in Optimizer Basal Culture Medium ((Invitrogen, USA)) containing interleukin-2 ((Novartis Pharma AG, Basel, Switzerland)) and then cultured in a flask at 37? C. for 10 days?14 days under 5% CO 2. Feeder layer or animal derived serum was never used in the entire process.

[0107] The NK cells were divided into normal medium group TGP medium group. In the normal medium group, approximately 20,000 NK cells, dispersed in ALyS 505 medium ((Cell Science & Technology Inst., Inc. Japan)) were placed in 2 mL sterile microcentrifuge tubes. In the TGP medium group, approximately 2,000 NK cells, dispersed in TGP medium were placed in 2 mL sterile microcentrifuge tubes. 1 g of freeze-dried TGP (Mebiol Gel, Mebiol Co., Ltd., Hiratsuka City) was dissolved in 10 mL of ALyS 505 medium, and the cells were dispersed in low-temperature sol state.

[0108] The normal medium group and TGP medium group were respectively divided into the normal gravity group and the microgravity group. The microgravity group was cultured at 37? C. for 6 hours in the microgravity mode ( 1/1000 G) in a microgravity environment cell culture equipment called Zeromo (CL-5000, manufactured by Kitagawa Iron Works, Ltd.), and the normal gravity group was cultured at 37? C. for 6 hours in normal environment.

[0109] Then, the viable cell population was measured in the collected NK cells, and measurement was conducted for the NO level and FACS analysis.

[0110] NO level was measured using Griess assay. Direct detection is extremely difficult as the half-life of the aqueous solution of NO is as short as 3-5 seconds, but the NO2 and NO3 oxides of NO are stable. Griess assay is the standard method of detecting NO2 in a culture supernatant by color reaction.

[0111] When comparing by absorbance in Griess assay, the NO level was (a) 0.090 under normal gravity and (b) 0.084 under microgravity in the normal medium. The NO level in TGP medium was (c) 0.126 under normal gravity and (d) 0.154 under microgravity. In other words, the NO level of NK cells had increased 1.7 times under the microgravity in TGP medium compared to the normal medium under normal gravity.

[0112] The cell population is the relative value, wherein the initial cell population is taken as 1. It was observed to be (a) 1.02 under normal gravity and (b) 1.32 under microgravity in normal medium, (c) 1.65 under normal gravity and (d) 1.27 under microgravity in TGP medium

TABLE-US-00001 TABLE 1 <Result of FACS analysis> (Example 1) Cell population CD56 CD16 CD3 Positive Positive Positive cell cell cell popula- popula- popula- tion tion tion Initial cell population 1 1 1 Normal medium Under normal gravity 0.87 0.70 1.11 (Cell population) Under microgravity 1.06 0.43 1.21 TGP medium Under normal gravity 0.66 0.15 1.03 (Cell population) Under microgravity 0.83 0.53 1.19

Example 2

[0113] The cell population, NO level and FACS analysis results were compared for cord blood mononuclear cells (UCBMNCs) cells cultured in normal medium under normal gravity (a) for 6 hours, and under microgravity (b), and those cultured in TGP medium under normal gravity (c) and under microgravity (d) for 6 hours.

[0114] UCBMNCs were isolated from cord blood banks in India, from cord blood not used in transplantation medicine. Isolation and culturing of the cells was carried out in GMP conformant class 10,000 clean room. The collected peripheral blood was centrifuged at room temperature at 3,000 rpm for 10 minutes, the supernatant plasma was discarded and the precipitated hemocyte pellets were transferred to another sterile centrifuge tube. Phosphate buffer (PBS) was mixed well with the hemocyte pellets placed in a sterile centrifuge tube in a capacitance ratio of 1:1. Ficoll solution (GE Healthcare Life Sciences, UK, Catalog no. 17030010) was overlaid on the blood cell PBS dispersion in a capacitance ratio of 2:1 (Example, 15 mL of Ficoll solution in 30 mL of blood cell dispersion). After centrifuging at 1,500 rpm for 30 minutes at room temperature, the umbilical cord blood mononuclear cell layer was transferred to another sterile container without disturbing the isolation layer, and PBS which is twice the amount of the bone marrow mononuclear cell layer was added. After centrifuging at 1,200 rpm for 10 minutes at room temperature, the supernatant was removed with a pipette. 30 ml of phosphate buffer was added to the precipitated cells and the mixture was gently stirred. Next, 20 ?l of the mixture was sampled and the cell population was measured. In the measurement of cell population, 20 ?l of Turk's staining solution was added to 20 ?l of the cell dispersion, 10 ?l was then sampled and the viable cell population was measured. After 30 ml of the cell dispersion was centrifuged at 1,200 rpm for 8 minutes at room temperature, the supernatant was removed with a pipette.

[0115] UCBMNCs cells were divided into normal medium group and TGP medium group. About 20000 UCBMNCs cells dispersed in IMDM ((Gibco?, Life Technologies Corp, USA)) medium were placed in the normal medium group in 2 mL sterile microcentrifuge tubes. In the TGP medium group, TGP medium dispersed with approximately 20,000 UCBMNCs cells was placed in 2 mL sterile microcentrifuge tubes. 1 g of freeze-dried TGP (Mebiol Gel, Mebiol Co., Ltd., Hiratsuka City) was dissolved in 10 mL of IMDM and the cells were dispersed at low-temperature sol state. The TGP solution was allowed to gel at room temperature, and then overlaid with commercial human serum. After that, the cells were cultured at 37? C. under 5% CO2 environment for 7-14 days.

[0116] Normal medium group and TGP medium group were divided into normal gravity group and microgravity group, respectively. The microgravity group was cultured at 37? C. for 6 hours in the microgravity mode ( 1/1000 G) with Zeromo, a microgravity environment cell culture equipment (CL-5000, manufactured by Kitagawa Iron Works, Co., Ltd.). The normal gravity group was cultured at 37? C. for 6 hours in normal environment.

[0117] After that, the collected UCBMNCs cells were measured for viable cell population and NO level, and the FACS analysis was measured.

[0118] The NO level was measured using DAR (diaminorhodamine) assay. DAR assay is a method of using a fluorescent dye to directly detect NO in cells or supernatant. When relatively compared by fluorescence intensity, the NO level in normal medium was (a) (1.75) under normal gravity and (b) 2.69 under microgravity. In the TGP medium it was (c) 2.35 under normal gravity and (d) 3.10 under microgravity. That is, compared to normal medium and under normal gravity, the NO level of UCBMNCs cells had increased 1.8 times in the TGP medium under microgravity.

[0119] The cell population is the relative value, wherein the initial cell population is taken as 1. In the normal medium, it was (a) 1.17 under normal gravity and (b) 1.29 under microgravity. In TGP medium, it was (c) 1.17 under normal gravity, and (d) 1.29 under microgravity.

TABLE-US-00002 TABLE 2 <Result of FACS analysis> (Example 2) Cell population CD34 Positive cell population Initial cell population 1 Normal medium Under normal gravity 4.40 (Cell population) Under microgravity 1.53 TGP medium Under normal gravity 2.29 (Cell population) Under microgravity 20.84

[0120] In other words, CD34 positive cell population of UCBMNCs cells had increased as much as 13.6 times in the TGP medium under microgravity compared to the normal medium microgravity.

Example 3

[0121] The NO level and FACS analysis result were compared for bone marrow mononuclear (BMMNCs) cells cultured in normal medium for 6 hours under normal gravity (a) and under microgravity (b), and those cultured in TGP medium for 6 hours under normal gravity (c) and microgravity (d). Bone marrow harvested from a human was placed in a sterile centrifuge tube and centrifuged at 3,000 rpm for 10 minutes at room temperature; the supernatant was then removed with a pipette. 15 ml of precipitated cells were dispersed by adding 15 ml of phosphate buffer (Manufactured by Life Technologies, USA, Catalog no. 70011-044), and 15 ml of Ficoll solution (manufactured by GE Healthcare Life Sciences, UK Catalog No. 17030010) and overlaid. After centrifuging at 1,500 rpm for 30 minutes at room temperature, the bone marrow mononuclear cell layer was transferred to another sterilization container without disturbing the isolation layer, and phosphate buffer solution, which is twice the amount of bone marrow mononuclear cell layer, was added. After centrifuging at 1,200 rpm for 10 minutes at room temperature, the supernatant was removed with a pipette. 30 ml of phosphate buffer was added to the precipitated cells, the mixture was gently stirred, 20 ?l of the mixture was sampled and the cell population was measured. In measuring the cell population, 20 ?l of Turk's stain solution was added to 20 ?l of the cell dispersion, 10 ?l was sampled and the viable cell population was measured. The cell dispersion was centrifuged at 1,200 rpm for 8 minutes at room temperature, and the supernatant was removed with a pipette.

[0122] Thereinafter, the same experiment as in Example 2 was carried out, and the NO level and FACS analysis were measured for the BMMNCs cells.

[0123] When NO levels were measured in DAR assay and relatively compared by the fluorescence intensity, the NO level in normal medium was (a) 0.95 under normal gravity and (b) 0.78 under normal gravity; in the TGP medium, it was (c) 0.87 under normal gravity and (d) 2.90 under microgravity. In other words, the NO level of UCBMNCs cells was found to have increased 3.1 times under TGP medium microgravity than under normal gravity in normal medium.

TABLE-US-00003 TABLE 3 <Result of FACS analysis> (Example 3) Cell population CD34 Positive cell population Initial cell population 1 Normal medium Under normal gravity 0.59 (Cell population) Under microgravity 1.71 TGP medium Under normal gravity 0.78 (Cell population) Under microgravity 1.57

[0124] That is, the CD34 positive cell population in BMMNCs cells had increased 2.7 times under TGP medium microgravity as compared to normal medium normal gravity.

INDUSTRIAL APPLICABILITY

[0125] As specifically explained above, here, it is possible to efficiently enhance the function of NK cells, and has substantial utility in the treatment of solid organ cancer, treatment of blood cancers, and in the treatment of patients who are bone marrow transplant recipients

[0126] This method of regeneration can be very effectively used as the method of cell recovery, restoration and revival in regenerative medicine.