Industrial preparations of natural killer (NK) cells and injections containing NK cells

Abstract

An industrial preparation of natural killer cells (NKs) is produced by: using umbilical cord blood and peripheral blood from legitimate sources as raw materials, obtaining stem cells by a method for extracting and separating karyocytes, or using FICOLL? or PERCOLL? density gradient media centrifugation to isolate and screen out karyocytes; diluting the above-mentioned karyocytes with cell culture medium, adding interferon, interleukin, CD3 antibody, and human albumin, loading them together into a bioreactor for perfusion culture, and then performing multiplication culture; the passage number of natural killer cells from multiplication culture is no less than 8, and the culture time is no less than 4 weeks; the markers of the natural killer cells obtained after the multiplication culture are CD3.sup.?\CD56.sup.+, CD16.sup.+, CD57.sup.+, and CD8.sup.+, wherein CD16.sup.+/CD56.sup.+?15%, CD3.sup.?/CD56.sup.+?50%, and CD8.sup.+/CD57.sup.+?8%; then preparing an injection with a certain concentration using the cell suspension obtained by above-mentioned method.

Claims

1. A method for industrially preparing a population of natural killer (NK) cells from umbilical cord blood and/or peripheral blood, the method comprising: obtaining human allogeneic karyocytes comprising stem cells from umbilical cord blood and/or peripheral blood by isolating the human allogeneic karyocytes using density gradient media centrifugation; diluting the human allogeneic karyocytes in cell culture medium, wherein the cell culture medium is RPMI 1640; adding gamma interferon (IFN?), interleukin (IL)-7, an anti-CD3 antibody, and human albumin, to the cell culture medium, loading the cell culture medium comprising the human allogeneic karyocytes, IFN?, the interleukin, the anti-CD3 antibody, and the human albumin into a bioreactor or perfusion incubator for multiplication culture; and performing multiplication culture in the cell culture medium comprising IFN?, the interleukin, the anti-CD3 antibody, and human albumin to culture NK cells from the human allogeneic karyocytes, wherein the time of the multiplication culture is no less than 4 weeks; thereby producing the population of NK cells, wherein at least 15% of the NK cells in the population are CD16.sup.+/CD56.sup.+, at least 50% of the NK cells in the population are CD3.sup.?/CD56.sup.+, and at least 8% of the NK cells in the population are CD8.sup.+/CD57.sup.+.

2. The method according to claim 1, wherein the cell culture medium is serum-free RPMI 1640.

3. The method according to claim 1, wherein the temperature for multiplication culture is 37? C.

4. The method according to claim 1, wherein the multiplication culture is carried out in the bioreactor.

5. The method according to claim 1, wherein human cord blood serum and/or human peripheral blood serum is also included in the cell culture medium loaded into the bioreactor.

6. The method according to claim 1, further comprising preparing a composition comprising a cell suspension of the NK cells from the multiplication culture.

7. The method according to claim 1, wherein no cell culture medium is added again for two days after loading the cell culture medium including the human allogeneic karyocytes, the IFN?, the IL-7, the anti-CD3 antibody, and the human albumin into the bioreactor or the perfusion incubator.

Description

DETAILED DESCRIPTION OF THE INVENTION

Example 1

(1) An umbilical cord blood sample (no less than 50 ml) from healthy donors was obtained from the umbilical cord blood bank, together with the traceable donor code, wherein the donors have neither genetic diseases nor the four infectious diseases including hepatitis B, hepatitis C, HIV/AIDS, and syphilis). For the sake of security, an additional umbilical cord blood sample of 1 ml was obtained and delivered to a third part for detecting hepatitis B, hepatitis C, HIV/AIDS, and syphilis, as well as ABO blood group and RH blood group, for archiving and future traceability.

(2) After identified as qualified, the donor umbilical cord blood was immediately used for the preparation of karyocytes. The separation and extraction of karyocytes from the umbilical cord blood were conducted using the Bone Marrow and Umbilical Cord Blood Cells Processing Kit (Ningxia Zhonglianda Biotech Co., Ltd) according to the manufacturer's instructions.

(3) During the separation, all the umbilical cord blood serum should be preserved and filtered through a 0.22 ?m filter for further use.

(4) The culture medium for natural killer cells is prepared as follows:

(5) Adding the umbilical cord blood serum preserved during the preparation of the karyocytes to serum-free medium GT-T551 in a ratio of 1:200 by weight, with a total volume of about 10000 ml.

(6) Additionally adding 0.1-0.5% (wt) human albumin (pharmaceutical grade), 20 U-200 U/ml (international unit) interleukin-2, 5-50 mg/ml CD3 monoclonal antibody, and 10 U-100 U/ml IFN-?; and

(7) Assaying the survival rate of the isolated and extracted umbilical cord blood karyocytes, performing cell counting to obtain the total number, and then adding the culture medium for natural killer cells in a concentration of 1?10.sup.6 /ml calculated based on cell number, wherein the total volume of the natural killer cells culture medium is about 100-150 ml, since typically 1?10.sup.8 karyocytes may be isolated and extracted from an umbilical cord blood sample, and 50 ?l sample was taken and assayed for DNA using microarray sequencing, and the assay result was compared with the assay result for DNA of the finished injections of natural killer cell.

(8) The induced natural killer cells culture medium containing the umbilical cord blood karyocytes was loaded into the Wave bioreactor (U.S. GE Corporation) for perfusion culture.

(9) Conditions: 37? C.?0.2? C. and 5% CO.sub.2.

(10) The culture medium is mixed well by built-in agitator, with a rotating speed of 8-12 times/min.

(11) No formulated natural killer cells culture medium was added in the first two days. From the third day, based on the cell growth speed and the calculated concentration of 1-5?10.sup.6/ml, a corresponding amount of natural killer cells culture medium was added, and the perfusate was removed in a ratio of 1/2 of the added cell medium until all the formulated natural killer cells culture medium ran out. The culture was maintained for 1-2 days until the cell concentration reached 1?10.sup.7/ml.

(12) Continuous culture was conducted for 4-6 weeks, and the culture was completed when the cell density reached or was close to 1?10.sup.7/ml, and the calculated total number of cells reached 1?10.sup.10. Alternatively, continuous culture was conducted 6 weeks before the culture was completed. All the cells were collected carefully, and washed twice with cell maintenance medium.

(13) [Assay]

(14) Assay steps should be performed first.

(15) A cell suspension of 1?10.sup.6/ml was prepared after the processes for culturing the natural killer cells and the finished products were testified as qualified.

(16) The cell suspension was sub-assembled into bags with a specification of 2?10.sup.7/20 ml or 5?10.sup.7/50 ml, and then 5% CO.sub.2 was added for sealing, thereby obtaining the injections of natural killer cell (cell medicine). These injections could be directly used by the clinician for treatment, of which the preservation and transportation must be conducted at a lower temperature (an optimum temperature of 4? C.).

(17) 1. Assay of the Cell Culture Procedure (Process Quality Control)

(18) The removed perfusate was assayed daily for bacterial endotoxin from the third day during the culture of natural killer cells (bacterial endotoxin?5 EU/ml).

(19) Three days before the end of the culture of natural killer cells, samples were collected daily for sterility test. The assay method used herein is described in the supplementary provisions of Chinese Pharmacopoeia 2010.

(20) A cell morphology test must be performed weekly during the culture of natural killer cells, for observing the karyotype and cell size, and determining whether the cell morphology is normal.

(21) 2. The Assay of the Finished Injection of Natural Killer Cell

(22) After the culture of the natural killer cells was completed, upon the concentration and washing of cells, the culture medium was sampled and assayed (bacterial endotoxin?5 EU/ml).

(23) The culture medium was sampled and assayed for mycoplasma, and the result should be negative. The assay method used herein is described in Part 6, Chapter 5 of National Guide to Clinical Laboratory Procedures, Third Edition, edited by Department of Medical Administration, Ministry of Health.

(24) The flow cytometry was used to assay the cell phenotype antigen of the natural killer cells injections. The test items and normal values were as follows: CD3.sup.?, CD56.sup.+, CD16.sup.+, CD57.sup.+, and CD8.sup.+, wherein CD16.sup.+/CD56.sup.+?15%, CD3.sup.?/CD56.sup.+?50%, and CD8.sup.+/CD57.sup.+?8%.

(25) The cell survival rate was assayed by using trypan blue staining method, wherein the product with the assay result of survival rate?96% was qualified, otherwise unqualified.

(26) 50 ?l of the product was assayed for DNA using microarray sequencing. The assay result was compared with the DNA of primary cells to determine if there was any variation: no DNA variation indicates that the product is normal and qualified. The assay results of this Example are shown in Table 1.

(27) TABLE-US-00001 TABLE 1 The assay results of NK cells qualified criteria No. test items that are designed found results 1 bacterial endotoxin ?5 EU/ml ?5 EU/ml qualified 2 mycoplasma negative negative qualified 3 CD16.sup.+/CD56.sup.+ ?15% 18% qualified 4 CD3.sup.-/CD56.sup.+ ?50% 80% qualified 5 CD8.sup.+/CD57.sup.+ 8% 8.5% qualified 6 cell survival rate ?96% 98% qualified 7 DNA assay no variation no variation qualified

(28) After the above items all pass the test, this batch of natural killer cell injections are confirmed as qualified medicines, and can be sub-assembled and labeled for delivery. The natural killer cell injections of each batch are required to be traceable, and the samples should be cryopreserved for more than five years.

(29) The above natural killer cells injections were used to treat the patients suspected of liver cancer metastasis after surgical removal of liver cancer, who has alpha-fetoprotein (AFP)>400 ng/ml (ELISA assay), the total number of leukocytes<3.6?10.sup.9/L, and other significantly decreased immune indexes. A 50 ml injection of natural killer cell with cell concentration of 1?10.sup.6/ml was intravenously injected once every other day, with a total of five times for a course of treatment.

(30) After a course of treatment was completed, the AFP level in 46% patients started to decrease at the first weekend, and the AFP level in 91.5% patients may be less than 100 ng/ml or close to normal level at the fourth weekend. Leukocyte level began to increase at the first weekend, and raised to normal level in 91% patients over two weeks (male: 3.97?9.15?10.sup.9/L, female: 3.69?9.16?10.sup.9/L). The general conditions of these patients improved expressly. The second and third courses of treatment were carried out at an interval of 3-6 months based on the specific states of the patients, and the two-year survival rate significantly increased by nearly 50%.

(31) Adverse reactions: after the intravenous injection of natural killer cell injections, low-grade fever occurs occasionally but the temperature rarely exceeds 37.6? C. Under such circumstances, physical cooling can be performed. The low-grade fever remains for one day before the patient spontaneously recovers. Occasional nausea and vomiting may also happen.

Example 2

(32) A 200 ml peripheral blood sample from healthy donors was obtained from center blood banks, wherein the sample has the assay report about four infectious diseases including hepatitis B, hepatitis C, HIV/AIDS and syphilis, together with the traceable donor code. For the sake of security, an additional blood sample of 1 ml was obtained and delivered to a third part for detecting hepatitis B, hepatitis C, HIV/AIDS, and syphilis, as well as ABO blood group and RH blood group, for archiving and future traceability.

(33) After identified as qualified, the donor peripheral blood was immediately used for the preparation of karyocytes. The separation and extraction of karyocytes from the peripheral blood were conducted using the Bone Marrow and Umbilical Cord Blood Cells Processing Kit (Ningxia Zhonglianda Biotech Co., Ltd) according to the manufacturer's instructions.

(34) During the separation, all the peripheral blood serum should be preserved and filtered through a 0.22 ?m filter for further use.

(35) The culture medium for natural killer cells is prepared as follows:

(36) Adding the peripheral blood serum preserved during the preparation of the karyocytes to serum-free medium GT-T551 in a ratio of 1:100 by weight, with a total volume of about 10000 ml.

(37) Additional adding the following components:

(38) 0.1% (wt) human albumin (pharmaceutical grade);

(39) 20 U/ml (international unit) interleukin-1;

(40) 5 mg/ml CD3 monoclonal antibody; and

(41) 10 U/ml IFN-?.

(42) Assaying the survival rate of the isolated and extracted peripheral blood karyocytes, performing cell counting to obtain the total number, and then adding the culture medium for natural killer cells in a concentration of 1?10.sup.6 /ml calculated based on cell number, wherein the total volume of the natural killer cells culture medium is about 200 ml, since typically 2?10.sup.8 karyocytes may be isolated and extracted from a conventional peripheral blood sample of 200 ml.

(43) The induced natural killer cells culture medium containing the peripheral blood karyocyte was loaded into the domestic industrial reaction kettle for perfusion culture. 50 ?l sample was taken and assayed for DNA using microarray sequencing, and the assay result was compared with the assay result for DNA of the finished injection of natural killer cell.

(44) Conditions: 37? C.?0.2? C. and 5% CO.sub.2.

(45) The culture medium is mixed well by built-in agitator, with a rotating speed of 8 times/min.

(46) No formulated natural killer cells culture medium was added in the first two days. From the third day, based on the cell growth speed and the calculated concentration of 1-5?10.sup.6/ml, a corresponding amount of natural killer cells culture medium was added, and the perfusate was removed in a ratio of 1/2 of the cell medium added until all the formulated natural killer cells culture medium ran out. The culture was maintained for 1-2 days until the cell concentration reached 1?10.sup.7/ml.

(47) Continuous culture was conducted for 4-6 weeks, and the culture was completed when the cell density reached or was close to 1?10.sup.7/ml, and the calculated total number of cells reached 1?10.sup.10. Alternatively, continuous culture was conducted 6 weeks before the culture was completed. All the cells were collected carefully, and washed twice with cell maintenance medium. The assay results of this Example are shown in Table 2.

(48) TABLE-US-00002 TABLE 2 The assay results of NK cells qualified criteria No. test items that are designed found results 1 bacterial endotoxin ?5 EU/ml ?5 EU/ml qualified 2 mycoplasma negative negative qualified 3 CD16.sup.+/CD56.sup.+ ?15% 17% qualified 4 CD3.sup.-/CD56.sup.+ ?50% 79% qualified 5 CD8.sup.+/CD57.sup.+ 8% 8.3% qualified 6 cell survival rate ?96% 97% qualified 7 DNA assay no variation no variation qualified

(49) A cell suspension of 1?10.sup.6/ml was prepared after the processes for culturing the natural killer cells and the finished products were testified as qualified.

(50) The cell suspension was sub-assembled into bags with a specification of 2?10.sup.7/20 ml or 5?10.sup.7/50 ml, and then 5% CO.sub.2 was added for sealing, thereby obtaining the injections of natural killer cell (cell medicine). These injections could be directly used by the clinician for treatment, of which the preservation and transportation must be conducted at a lower temperature (an optimum temperature of 4? C.).

(51) [Assay]

(52) The assays of cell culture procedure and the finished injection of natural killer cell are the same as those in Example 1.

(53) The finished injection of natural killer cell was used to treat the patients suspected of metastasis after surgical removal of uterine cancer, who has clinical carcinoembryonic antigen (CEA)>20 ng/ml (ELISA assay) and the significantly lower total number of leukocytes than normal level. A 50 ml injection of natural killer cell with cell concentration of 1?10.sup.6/ml was intravenously injected once every other day, with a total of five times for a course of treatment.

(54) Seven days after the uterine cancer patients were intravenously injected with the injections of natural killer cell, the CEA level in 41% patients started to decrease, and the CEA level in 95% patients was below 10 ng/ml or close to normal level of 3 ng/ml at the fourth weekend. Leukocyte level began to increase at the first weekend, and raised to normal level in 90% patients over two weeks (male: 3.97?9.15?10.sup.9/L, female: 3.69?9.16?10.sup.9/L). The general conditions of these patients improved expressly. The second and third courses of treatment were carried out at an interval of 3-6 months based on the specific states of the patients.

(55) Adverse reactions: after the intravenous injection of natural killer cell injections, low-grade fever occurs occasionally but the temperature rarely exceeds 37.6? C. Under such circumstances, physical cooling can be performed. The low-grade fever remains for one day before the patient spontaneously recovers. Occasional nausea and vomiting may also happen.

Example 3

(56) An umbilical cord blood sample (no less than 50 ml) from healthy donors was obtained from the umbilical cord blood bank, together with the traceable donor code, wherein the donors have neither genetic diseases nor the four infectious diseases including hepatitis B, hepatitis C, HIV/AIDS, and syphilis). For the sake of security, an additional umbilical cord blood sample of 1 ml was obtained and delivered to a third part for detecting hepatitis B, hepatitis C, HIV/AIDS, and syphilis, as well as ABO blood group and RH blood group, for archiving and future traceability.

(57) After identified as qualified, the donor umbilical cord blood was immediately used for the preparation of karyocytes. FICOLL? or PERCOLL? density gradient media centrifugation was used to sort out mononuclear cells.

(58) 1. The whole blood of umbilical cord blood was added into a 50 ml centrifuge tube, and centrifuged at 800?g for 5 minutes. All the umbilical cord blood serum was collected carefully, and filtered and sterilized through a 0.22 ?m filter for further use.

(59) 2. The red blood cells and white blood cells deposited at the bottom of the centrifuge tube were dissolved with PBS solution, and the total volume was identical with that of this umbilical cord blood.

(60) 3. Monocytes (karyocytes) were sorted out from the dissolved red blood cells and white blood cells using FICOLL? or PERCOLL? density gradient media centrifugation, and calculated for the total cell number.

(61) The culture medium for natural killer cells is prepared as follows:

(62) Adding the umbilical cord blood serum preserved during the preparation of the karyocytes to serum-free medium RMP I-1640 in a ratio of 1:200, with a total volume of around 10000 ml.

(63) Additionally adding 0.5% (wt) human albumin (pharmaceutical grade), 200 U/ml (international unit) interleukin-7, 50 mg/ml CD3 monoclonal antibody, and 100 U/ml IFN-?.

(64) Assaying the survival rate of the isolated and extracted umbilical cord blood karyocytes, performing cell counting to obtain the total number, and then adding the culture medium for natural killer cells in a concentration of 1?10.sup.6 /ml calculated based on cell number, wherein the total volume of the natural killer cells culture medium is about 100-150 ml, since typically 1?10.sup.8 karyocytes may be isolated and extracted from an umbilical cord blood sample, and 50 ?l sample was taken and assayed for DNA using microarray sequencing, and the assay result was compared with the assay result for DNA of the finished injections of natural killer cell.

(65) The induced natural killer cells culture medium containing the umbilical cord blood karyocytes was loaded into the Wave bioreactor (U.S. GE Corporation) for perfusion culture.

(66) Conditions: 37? C.?0.2? C. and 5% CO.sub.2.

(67) The culture medium is mixed well by built-in agitator, with a rotating speed of 8-12 times/min.

(68) No formulated natural killer cells culture medium was added in the first two days. From the third day, based on the cell growth speed and the calculated concentration of 1-5?10.sup.6/ml, a corresponding amount of natural killer cells culture medium was added, and the perfusate was removed in a ratio of 1/2 of the cell medium added until all the formulated natural killer cells culture medium ran out. The culture was maintained for 1-2 days until the cell concentration reached 1?10.sup.7/ml.

(69) Continuous culture was conducted for 4-6 weeks, and the culture was completed when the cell density reached or was close to 1?10.sup.7/ml, and the calculated total number of cells reached 1?10.sup.10. Alternatively, continuous culture was conducted 6 weeks before the culture was completed. All the cells were collected carefully, and washed twice with cell maintenance medium. The assay results of this Example are shown in Table 3.

(70) TABLE-US-00003 TABLE 3 The assay results of NK cells qualified criteria No. test items that are designed found results 1 bacterial endotoxin ?5 EU/ml ?5 EU/ml qualified 2 mycoplasma negative negative qualified 3 CD16.sup.+/CD56.sup.+ ?15% 17% qualified 4 CD3.sup.-/CD56.sup.+ ?50% 81% qualified 5 CD8.sup.+/CD57.sup.+ 8% 8.2% qualified 6 cell survival rate ?96% 99% qualified 7 DNA assay no variation no variation qualified

(71) A cell suspension of 1?10.sup.6/ml was prepared after the processes for culturing the natural killer cells and the finished products were testified as qualified.

(72) The cell suspension was sub-assembled into bags with a specification of 2?10.sup.7/20 ml or 5?10.sup.7/50 ml, and then 5% CO.sub.2 was added for sealing, thereby obtaining the injections of natural killer cell (cell medicine). These injections could be directly used by the clinician for treatment, of which the preservation and transportation must be conducted at a lower temperature (an optimum temperature of 4? C.).

(73) [Assay]

(74) The assays of cell culture procedure and the finished injection of natural killer cell are the same as those in Example 1.

(75) The finished injection of natural killer cell was used to treat the patients having metastasis after surgical removal of gastric cancer, who has clinical carcinoembryonic antigen (CEA)>20 ng/ml (ELISA assay) and the significantly lower total number of leukocytes than normal level. A 50 ml injection of natural killer cell with cell concentration of 1?10.sup.6/ml was intravenously injected once every other day, with a total of three times for a course of treatment.

(76) Seven days after the gastric cancer patients were intravenously injected with the injection of natural killer cell, the CEA level in 52% patients started to decrease, and the CEA level in 90% patients was below 10 ng/ml or close to normal level of 3 ng/ml at the fourth weekend. Leukocyte level began to increase at the first weekend, and raised to normal level in 89% patients over two weeks (male: 3.97?9.15?10.sup.9/L, female: 3.69?9.16?10.sup.9/L). The general conditions of these patients improved expressly. The second and third courses of treatment were carried out at an interval of 3-6 months based on the specific states of the patients.

(77) Adverse reactions: after the intravenous injection of natural killer cell injections, low-grade fever occurs occasionally but the temperature rarely exceeds 37.6? C. Under such circumstances, physical cooling can be performed. The low-grade fever remains for one day before the patient spontaneously recovers. Occasional nausea and vomiting may also happen.

Example 4

(78) A 200 ml peripheral blood sample from healthy donors was obtained from center blood banks, wherein the sample has the assay report about four infectious diseases including hepatitis B, hepatitis C, HIV/AIDS and syphilis, together with the traceable donor code. For the sake of security, an additional blood sample of 1 ml was obtained and delivered to a third part for detecting hepatitis B, hepatitis C, HIV/AIDS, and syphilis, as well as ABO blood group and RH blood group, for archiving and future traceability.

(79) After identified as qualified, the donor peripheral blood was immediately used for the preparation of karyocytes. The separation and extraction of karyocytes from the peripheral blood were conducted using the Bone Marrow and Umbilical Cord Blood Cells Processing Kit (Ningxia Zhonglianda Biotech Co., Ltd) according to the manufacturer's instructions.

(80) 1. The whole blood of peripheral blood was added into a 50 ml centrifuge tube, and centrifuged at 800?g for 5 minutes. All the peripheral blood serum was collected carefully, and filtered through a 0.22 ?m filter for further use.

(81) 2. The red blood cells and white blood cells deposited at the bottom of the centrifuge tube were dissolved with PBS solution, and the total volume was identical with that of this peripheral blood, i.e. 200 ml.

(82) 3. Monocytes (karyocytes) were sorted out from the dissolved red blood cells and white blood cells using FICOLL? or PERCOLL? density gradient media centrifugation, and calculated for the total cell number.

(83) The culture medium for natural killer cells is prepared as follows:

(84) Adding the peripheral blood serum preserved during the preparation of the karyocytes to serum-free medium RMP I-1640 in a ratio of 1:100, with a total volume of around 10000 ml.

(85) Additionally adding 0.5% (wt) human albumin (pharmaceutical grade), 100 U/ml (international unit) interleukin-7, 20 mg/ml CD3 monoclonal antibody, and 100 U/ml IFN-?.

(86) Assaying the survival rate of the isolated and extracted peripheral blood karyocytes, performing cell counting to obtain the total number, and then adding the culture medium for natural killer cells in a concentration of 1?10.sup.6 /ml calculated based on cell number, wherein the total volume of the natural killer cells culture medium is about 200 ml, since typically 2?10.sup.8 karyocytes may be isolated and extracted from a conventional peripheral blood sample of 200 ml.

(87) The induced natural killer cells culture medium containing the peripheral blood karyocyte was loaded into the bioreactor for perfusion culture.

(88) Conditions: 37? C.?0.2? C. and 5% CO.sub.2.

(89) The culture medium is mixed well by built-in agitator, with a rotating speed of 8-12 times/min.

(90) No formulated natural killer cells culture medium was added in the first two days. From the third day, based on the cell growth speed and the calculated concentration of 1-5?10.sup.6/ml, a corresponding amount of natural killer cells culture medium was, and the perfusate was removed in a ratio of 1/2 of the cell medium added until all the formulated natural killer cells culture medium ran out. The culture was maintained for 1-2 days until the cell concentration reached 1?10.sup.7/ml.

(91) Continuous culture was conducted for 4-6 weeks, and the culture was completed when the cell density reached or was close to 1?10.sup.7/ml, and the calculated total number of cells reached 1?10.sup.10. Alternatively, continuous culture was conducted 6 weeks before the culture was completed. All the cells were collected carefully, and washed twice with cell maintenance medium.

(92) A cell suspension of 1?10.sup.6/ml was prepared after the processes for culturing the natural killer cells and the finished products were testified as qualified.

(93) The cell suspension was sub-assembled into bags with a specification of 2?10.sup.7/20 ml or 5?10.sup.7/50 ml, and then 5% CO.sub.2 was added for sealing, thereby obtaining the injections of natural killer cell (cell medicine). These injections could be directly used by the clinician for treatment, of which the preservation and transportation must be conducted at a lower temperature (an optimum temperature of 4? C.).

(94) [Assay]

(95) The assays of cell culture procedure and the finished injection of natural killer cell are the same as those in Example 1.

(96) The finished injection of natural killer cell was used to treat the patients having metastasis after surgical removal of gastric cancer, who has clinical carcinoembryonic antigen (CEA)>20 ng/ml (ELISA assay) and the significantly lower total number of leukocytes than normal level. A 50 ml injection of natural killer cell with cell concentration of 1?10.sup.6/ml was intravenously injected once every other day, with a total of three times for a course of treatment.

(97) Seven days after the gastric cancer patients were intravenously injected with the injection of natural killer cell, the CEA level in 52% patients started to decrease, and the CEA level in 90% patients was below 10 ng/ml or close to normal level of 3 ng/ml at the fourth weekend. Leukocyte level began to increase at the first weekend, and raised to normal level in 89% patients over two weeks (male: 3.97?9.15?10.sup.9/L, female: 3.69?9.16?10.sup.9/L). The general conditions of these patients improved expressly. The second and third courses of treatment were carried out at an interval of 3-6 months based on the specific states of the patients.

(98) Adverse reactions: after the intravenous injection of natural killer cell injections, low-grade fever occurs occasionally but the temperature rarely exceeds 37.6? C. Under such circumstances, physical cooling can be performed. The low-grade fever remains for one day before the patient spontaneously recovers. Occasional nausea and vomiting may also happen.

(99) The present invention solves the problem as to the conventional time-consuming process for preparing natural killer cells or CIK cells in clinical, and allows the clinician to conveniently use the injection of natural killer cell (pharmaceutical grade) anytime and anywhere, so as to treat the tumor patients as early as possible. In the meantime, the present invention enables industrial production of natural killer cells such that natural killer cell therapy technology develops from a clinical technology into the medicine in form of natural killer cells injections, realizing medicalization of the cells. Thus, the present invention sets a precedent for the industrial production of cell medicine, helps our country gain a place in the field of cell therapy for developing cell medicine with intellectual property, and make the cell medicine with intellectual property penetrate the international market.