CN105052895A - LAK cell preservation solution, preparation method thereof and LAK cell preservation method - Google Patents
LAK cell preservation solution, preparation method thereof and LAK cell preservation method Download PDFInfo
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- CN105052895A CN105052895A CN201510557439.9A CN201510557439A CN105052895A CN 105052895 A CN105052895 A CN 105052895A CN 201510557439 A CN201510557439 A CN 201510557439A CN 105052895 A CN105052895 A CN 105052895A
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Abstract
The invention relates to an LAK cell preservation solution, a preparation method thereof and a method for preserving LAK cells. The LAK cells can be stored at a temperature not higher than normal temperature by preparing LAK cells into a cell preparation using any one of the LAK cell preserving fluids described above. The LAK cell preservation solution can effectively maintain the activity of LAK cells; the LAK cell preservation solution is used for preserving the LAK cells, and the cell activity is maintained to be more than 90% after the LAK cells are preserved for 12 hours at normal temperature or 72 hours at 4 ℃; when the cell viability is maintained above 90%, the killing activity of LAK cells and the expression of surface markers are not changed significantly.
Description
Technical field
The present invention relates to field of cell culture, particularly relate to a kind of method of LAK cell-preservation liquid and preparation and preservation LAK cell.
Background technology
Wreck along with aging aggravation, ecotope, unhealthy life style and food-safety problem show especially, China's tumor incidence continues to rise for many years, has become a public health problem and even social concern that must pay much attention to.Estimate according to the report of world's cancer, within 2012, Cancer in China number of the infected is 306.5 ten thousand, accounts for 1/5th of whole world morbidity; Number of cancer deaths is 220.5 ten thousand, accounts for global number of cancer deaths's 1/4th.Current China cancer morbidity, lethality are sustainable growth trend, and China's cancer morbidity is close to world standard, but lethality is higher than world standard.More severe, this impetus is not effectively contained.20 years from now on, the morbidity number of China's cancer and death toll also will continue to rise: according to IARC's prediction, if do not adopted an effective measure, the year two thousand twenty is counted in China's pathogenesis of cancer number and death will rise to 4,000,000 people and 3,000,000 people; The year two thousand thirty will rise to 5,000,000 people and 3,500,000 people.
For a long time, the treatment of tumour generally all adopts operation, chemotherapy and radiation three kinds of Therapeutic mode.Along with the development of bioscience, biological therapy becomes the 4th kind of Therapeutic mode of oncotherapy.Biological therapy refers to the methods for the treatment of utilizing the immune system of human body self to kill and remove tumour cell.This method safety is effective, and a tumor killing cell, does not injure normal structure, and side effect is very little.Biological therapy can also alleviate the side effect of radiation and chemotherapy, strengthens patient to the susceptibility of other treatment and tolerance.LAK cell therapy is a kind of effective antitumour Biotherapy method, is the methods for the treatment of LAK cell of Activation In Vitro and amplification being inputed to patient after continuing.LAK cell (Lymphokine-activatedkiller) i.e. Tumor-infiltrating lymphocytes is that a kind of peripheral blood lymphocyte activates 3 ~ 5 days through lymphokine proleulzin (IL-2) and the killer cell increased as having wide spectrum Synergistic action in vitro.Research shows that LAK cell and IL-2 share, and (IL-2/LAK system adoptive immunotherapy) is more effective than alone IL-2, because the LAK cell activated through IL-2 still needs IL-2 could maintain its killing activity after inputting human body.IL-2/LAK system adoptive immunotherapy is be evaluated as one of large great scientific achievement eighties ten in 20th century, it is remarkable especially to metastatic renal cell cancer, black cancer, colon cancer and non_hodgkin lymphoma patient curative effect, also the metastasis (metastases) at the places such as the lung of other types tumour, liver, bone, skin can be made to disappear, and the oncocyte in circulation is disappeared.
Multiplication capacity, the killing activity of cultural method, cultivating system and the LAK cell studying LAK cell are only absorbed in the domestic and international research to LAK cell therapy at present mostly, rarely have research and report to the store method of the LAK cell preparation of clinical treatment level.In the prior art, the conserving liquid of LAK cell is all the physiological saline containing human serum albumins, as added the human serum albumins of 1% in physiological saline, the LAK cell preparation that this conserving liquid is preserved will decline more than the vigor of 24 hour cells more than 2 hours or under the condition of 4 DEG C at normal temperatures, is not suitable for long-distance transport or the preservation of LAK cell.
Summary of the invention
In view of this, be necessary, for the LAK cell problem that cytoactive declines in existing conserving liquid, to provide a kind of method of new LAK cell-preservation liquid and preparation and preservation LAK cell.
The invention provides a kind of LAK cell-preservation liquid, comprise human serum albumins, liquaemin, GL-B and preservation basal liquid, described preservation basal liquid is compound electrolyte solution.
Preferably, LAK cell-preservation liquid, is made up of the following component of following quality percentage by volume:
Human serum albumins 1-5%,
Liquaemin 1-10%,
GL-B 1-5%,
Compound electrolyte solution surplus.
Preferably, described LAK cell-preservation liquid is made up of the following component of following quality percentage by volume:
Human serum albumins 1-2%,
Heparin sodium aqua 3-8%,
GL-B 2-3%,
Compound electrolyte solution surplus.
Preferably, described LAK cell-preservation liquid is made up of the following component of following quality percentage by volume:
Human serum albumins 1%,
Heparin sodium aqua 5%,
GL-B 2%,
Compound electrolyte solution surplus.
Preferably, described compound electrolyte solution comprises sodium chloride, gluconic acid sodium salt, sodium acetate, potassium chloride and magnesium chloride.More preferably, described compound electrolyte solution is made up of the following component of following quality percentage by volume: sodium chloride 5.26%, gluconic acid sodium salt 5.02%, sodium acetate 3.68%, potassium chloride 0.37%, magnesium chloride 0.30%, water surplus.
The present invention also provides the preparation method of above-mentioned LAK cell-preservation liquid, with a small amount of compound electrolyte solubilize liquaemin and GL-B, then adds human serum albumins wherein, mixes and obtain LAK cell-preservation liquid of the present invention after supplying compound electrolyte solution.
The present invention also provides a kind of store method of LAK cell, uses the LAK cell-preservation liquid described in above-mentioned any one that LAK cell is made cell preparation, preserves in the following temperature of normal temperature.
Preferably, in described cell preparation, LAK cell concentration is 1 × 10
7/ mL.
Preferably, preservation condition is within normal temperature is preserved 12 hours.
Preferably, preservation condition is that 4 DEG C of preservations are within 72 hours.
Compared with prior art, the present invention has following beneficial effect:
LAK cell-preservation liquid component of the present invention is few, adds human serum albumins, liquaemin and GL-B in compound electrolyte solution, can direct feedback, each component and stable content thereof, and with low cost, conserving liquid is safe, without any side effects;
LAK cell-preservation liquid of the present invention effectively can maintain the vigor of LAK cell; Use LAK cell-preservation liquid of the present invention to preserve LAK cell, 12 hours later cell vigor of preserving at normal temperatures maintain more than 90%, or preserve after 72 hours under 4 DEG C of conditions, and cell viability also maintains about 90%;
LAK cell-preservation liquid of the present invention does not affect the killing activity of LAK cell and the expression of surface marker; When in LAK cell-preservation liquid of the present invention, cell viability maintains more than 90%, the killing activity of LAK cell does not have marked change, and the expression of surface marker is optimum;
LAK cell-preservation liquid preparation of the present invention simply, can be prepared in a large number at short notice, be produced on a large scale, and uses LAK cell-preservation liquid of the present invention to preserve the method for LAK cell also simply, just can preserve hand over for a long time in the following temperature of normal temperature, easy to utilize.
Accompanying drawing explanation
Fig. 1 is the LAK cellular morphology figure that in embodiment 5, PBMC obtains after cultivating 14 days, multiplication factor 400 times.
Fig. 2 is flow cytometer detection figure in effect example 3.
Embodiment
In order to better the present invention is described, be described further below in conjunction with the drawings and specific embodiments.In LAK cell-preservation liquid of the present invention, agents useful for same or instrument all can be buied by market, and the detection method of use is all known in the art, does not repeat them here.
The key instrument used in the present invention is respectively purchased from following company: cell culture incubator purchased from German MEMMERT company, inverted microscope purchased from Fujian MOTIC company, flow cytometer purchased from American BD company, microplate reader purchased from American Mei Liai company.
The main agents used in the present invention is respectively purchased from following company: Human Seroalbumin is purchased from Austrian Baxter Int, and wherein the quality percentage by volume of human serum albumins is 20%.Liquaemin (powder) cures Pharmaceuticals Ltd purchased from Guangzhou Zhong Shan.GL-B (freeze-dried powder) cures Pharmaceuticals Ltd purchased from Guangzhou Zhong Shan.Multiple electrolytes injection cures Pharmaceuticals Ltd purchased from Guangzhou Zhong Shan; Often liter of compound electrolyte solution contains sodium chloride 5.26g, gluconic acid sodium salt 5.02g, sodium acetate 3.68g, potassium chloride 0.37g, magnesium chloride 0.30g.
In the present invention, human serum albumins can prevent cell aggregation agglomerating; Liquaemin has blood coagulation resisting function, can prevent blood coagulation; GL-B can improve immunity of organisms, at cancer patient through radiotherapy, chemotherapy immunity of organisms are impaired, itself and radiotherapy, Chemotherapy plus is used the object that can reach cure diseases.
Compound electrolyte solution is compatible with blood constituent, and the LAK cell therefore using LAK cell-preservation liquid of the present invention to preserve directly feeds back, and does not need to cultivate or carry out other process again, simplifies use procedure, saves time.
embodiment 1, LAK cell-preservation liquid and preparation method thereof
With a small amount of compound electrolyte solution, 8g liquaemin powder and 2.5g GL-B freeze-dried powder are dissolved, add the Human Seroalbumin of 5mL mass percent 20% more wherein, add compound electrolyte solution and mix to 100mL and obtain LAK cell-preservation liquid of the present invention.Each constituent mass percentage by volume is in the present embodiment: human serum albumins 1%, liquaemin 8%, GL-B 2.5%, and surplus is compound electrolyte solution.
embodiment 2, LAK cell-preservation liquid and preparation method thereof
With a small amount of compound electrolyte solution, 5g liquaemin powder and 2g GL-B freeze-dried powder are dissolved, add the Human Seroalbumin of 5mL mass percent 20% more wherein, add compound electrolyte solution and mix to 100mL and obtain LAK cell-preservation liquid of the present invention.Each constituent mass percentage by volume is in the present embodiment: human serum albumins 1%, liquaemin 5%, GL-B 2%, and surplus is compound electrolyte solution.
embodiment 3, LAK cell-preservation liquid and preparation method thereof
With a small amount of compound electrolyte solution, 3g liquaemin powder and 5g GL-B freeze-dried powder are dissolved, add the Human Seroalbumin of 25mL mass percent 20% more wherein, add compound electrolyte solution and mix to 100mL and obtain LAK cell-preservation liquid of the present invention.Each constituent mass percentage by volume is in the present embodiment: human serum albumins 5%, liquaemin 3%, GL-B 5%, and surplus is compound electrolyte solution.
embodiment 4, LAK cell-preservation liquid and preparation method thereof
With a small amount of compound electrolyte solution, 1g liquaemin powder and 3g GL-B freeze-dried powder are dissolved, add the Human Seroalbumin of 20mL mass percent 20% more wherein, add compound electrolyte solution and mix to 100mL and obtain LAK cell-preservation liquid of the present invention.Each constituent mass percentage by volume is in the present embodiment: human serum albumins 4%, liquaemin 1%, GL-B 3%, and surplus is compound electrolyte solution.
The store method of embodiment 5, LAK cell
Use the LAK cell preparation that above-mentioned LAK cell is made by embodiment of the present invention 1-4 arbitrary LAK cell-preservation liquid, cell density is 1 × 10
7/ mL, cell viability is 98.6%.Above-mentioned LAK cell preparation is preserved under normal temperature and 4 DEG C of conditions.
Adopt in the present embodiment and prepare LAK cell with the following method: adopt Ficoll parting liquid to use sepmate centrifuge tube (purchased from STEMCELL company) separating peripheral blood mononuclear cells (PBMC) in 20mL peripheral blood; PBMC X-VIVO15 culture fluid is carried out resuspended, according to 5-10 × 10
5the cell density of individual/mL is seeded in T75 blake bottle, adds the IL-2 of OKT-3 and 100-1000U/mL of 10-50ng/mL, puts into 37 DEG C, the CO2gas incubator of 5% cultivates; Examine under a microscope the form of cell every day, carry out cell counting every three days, and according to 1 × 10
6the cell density of/mL adds the IL-2 of OKT-3,100-1000U/mL of a certain amount of X-VIVO15 culture fluid and 10-50ng/mL; Cultivate after 14 days, cell density is about 2 × 10
6/ mL, collects LAK cell.
Fig. 1 is the aspect graph of cultivation LAK cell after 14 days.LAK cellular morphology rule as seen from Figure 1, core is large, and circular, endochylema is few, is the LAK cell of maturation.
effect example 1, LAK cell viability effect
The vigor using the LAK cell that LAK cell-preservation liquid is preserved in embodiment 1-4 is detected respectively at 2h, 4h, 8h, 12h, 24h, 48h and 72h.Arrange following comparative example, in each comparative example except the composition of conserving liquid is different from content and the present invention, other conditions are identical simultaneously.
Comparative example 1 adopts the physiological saline containing mass percent 1% human serum albumins as conserving liquid.
The conserving liquid that comparative example 2 adopts comprises albumin, glucose, vitamin C and preservation basal liquid; Preservation basal liquid is volume ratio is the Multiple electrolytes injection of 1:1 and the mixture of Amino Acid Compound Injection.Described albuminous concentration expressed in percentage by volume is 1%, and the concentration expressed in percentage by volume of described glucose is 5%, and described ascorbic concentration expressed in percentage by volume is 0.5%.Described Multiple electrolytes injection is identical with the method electrolyte solution used in the present invention.
Amino Acid Compound Injection in comparative example 2 is that the component of Amino Acid Compound Injection is by 18 seed amino acids and sorbierite formulated sterile water solution: every 500mL contains: L-PROLINE (C
5h
9nO
2)0.50g; Serine (C
3h
7nO
3) 0.50g; ALANINE (C
3h
7nO
2) 1.00g; ILE (C
6h
13nO
2) 1.76g; L-Leu (C
6h
13nO
2) 2.45g; L-ASPARTIC ACID (C
4h
7nO
4) 1.25g; TYR (C
9h
11nO
3) 0.13g; Pidolidone (C
5h
9nO
4) 0.38g; L-Phe (C
9h
11nO
2) 2.67g; L-arginine hydrochloride (C
6h
14n
4o
2hCl) 2.50g; L lysine HCL (C
6h
14n
2o
2hCl) 2.15g; Valine (C
5h
11nO
2) 1.80g; L-threonine (C
4h
9nO
3) 1.25g; L-Histidine hydrochloride (C
6h
9n
3o
2hClH
2o) 1.25g; L-Trp (C
11h
12n
2o
2) 0.45g; L-Methionine (C
15h
11nO
2s) 1.13g; CYSTINE (C
6h
12n
2o
4s
2) 0.05g; Glycine (C
2h
5nO
2) 3.80g; Sorbierite (C
6h
14o
6) 25.00g; Sodium hydrogensulfite (NaHSO
3) 0.25g, auxiliary material is water for injection.
In embodiment 1-4 and comparative example, conserving liquid preserves the cell viability testing result of LAK cell in table 1.
Table 1LAK cell viability testing result
As shown in Table 1, compared with comparative example, LAK cell-preservation liquid of the present invention effectively can maintain the vigor of LAK cell.In comparative example at 4 DEG C more than 24 hours or at normal temperatures more than 2 hours, the vigor of LAK cell will obviously decline.Use LAK cell-preservation liquid of the present invention to preserve LAK cell, 12 hours later cell vigor of preserving at normal temperatures are 92-94%, and in average specific comparative example 2, the vigor of LAK cell is high by 3%; 12 hours later cell vigor of preserving under 4 DEG C of conditions are 95.33%, 72 hours later cell vigor of preserving are about 90%, high by 13.6% to LAK cell viability in comparative example 2, therefore, the LAK cell viability using LAK cell-preservation liquid of the present invention to preserve declines slowly, is applicable to preserve LAK cell for a long time.
effect example 2, LAK cell killing activity effect
To using the LAK cell that in embodiment 1, comparative example 1 and comparative example 2, conserving liquid is preserved, preserving at 4 DEG C after 72 hours, detecting the killing activity of LAK cell.The killing activity of the LAK cell that direct-detection is directly prepared with method described in embodiment 5 is comparative example 3.The cell pyrolysis liquid used in the present embodiment is 9%TritonX-100, purchased from PROMEGA; Substrate solution is SubstrateMix, purchased from PROMEGA; Stop buffer is 1Maceticacid.
During bed board, each experimental port and contrast arrange as follows:
Experimental port: the bed board carrying out target cell (K562) and effector cell's (LAK cell) by effect target than 40:1,20:1 and 10:1.Wherein, the concentration of LAK cell is respectively 4 × 10
6/ mL, 2 × 10
6/ mL, 1 × 10
6/ mL, every hole 100 μ L, often organize 3 multiple holes; K562 concentration is 1 × 10
5/ mL, every hole 100 μ L, often organize 3 multiple holes.
Effector cell's Spontaneous release hole: 4 × 10
6/ mL, 2 × 10
6/ mL, 1 × 10
6/ mL, every hole 100 μ L, often organize 3 multiple holes, every hole adds 100 μ L medium.
The maximum release aperture of target cell: 1 × 10
5the target cell of/mL, every hole 100 μ L, often organize 3 multiple holes, every hole adds 100 μ L medium, in 37 DEG C, 5% CO2gas incubator hatches the every hole of front 45min and adds 20 μ L cell pyrolysis liquids.
Target cell Spontaneous release hole: 1 × 10
5the target cell of/mL, every hole 100 μ L, often organize 3 multiple holes, every hole adds 100 μ L medium.
Culture fluid blank: every hole 200 μ L culture fluid, often organizes 3 multiple holes.
Volume corrects contrast: every hole 200 μ L culture fluid, often organizes 3 multiple holes, in 37 DEG C, 5% CO2gas incubator hatches the every hole of front 45min and adds 20 μ L cell pyrolysis liquids.
By centrifugal for culture plate 250g 4min after bed board, be placed in 37 DEG C, 5% CO2gas incubator hatches 4h.45min before cultivation terminates, takes out culture plate, adds 20 μ L cell pyrolysis liquids, the centrifugal 4min of 250g in target cell maximum release group and the every hole of volume correction group, takes out after continuing to cultivate 45min.
Carry out LDH (lactate dehydrogenase) to measure, concrete steps are as follows: the centrifugal 4min of 250g, and every hole sucking-off 50 μ L supernatant is transferred in another 96 new orifice plates, and every hole adds substrate solution 50 μ L, and room temperature lucifuge hatches 30min.Every hole adds 50 μ L stop buffers, is broken up by pigment granule with oscillator, measures the absorbance at 490 wavelength places.
The absorbance average of experimental group, target cell LDH Spontaneous release group and effector cell LDH Spontaneous release group deducts culture fluid blank group absorbance average, obtains corrected value.The absorbance average of the maximum release group of target cell LDH deducts volume correction group absorbance average, obtains corrected value.
Killing activity is calculated as follows:
Activity=(A-E-T)/(Tmax-T) × 100% ... (1)
A: experimental group absorbance corrected value,
E: effector cell's Spontaneous release hole absorbance corrected value,
T: target cell Spontaneous release hole absorbance corrected value,
Tmax: target cell maximum release group absorbance corrected value.
In the LAK cell that embodiment 1, comparative example 1-2 conserving liquid are preserved and comparative example 3, the killing activity of LAK cell is as shown in table 2.
Table 2 killing activity result
| Group | 40:1 | 20:1 | 10:1 |
| Embodiment 1 | 75.0％ | 48.2％ | 29.1％ |
| Comparative example 1 | 61.3％ | 42.5％ | 18.4％ |
| Comparative example 2 | 65.5％ | 42.86％ | 21.4％ |
| Comparative example 3 | 76.01％ | 49.2％ | 29.08％ |
As shown in Table 2, the LAK cell killing activity using the LAK cell-preservation liquid in embodiment 1 to preserve is substantially more constant than its killing activity during 40-10:1 at effect target, close with the activity without frozen LAK cell.Use the LAK cell killing activity of the preservation of the LAK cell-preservation liquid in embodiment 1 at effect target than being all significantly higher than the killing activity that in comparative example 1-2, conserving liquid is preserved during 40-10:1.Imitating target than during for 40:1, the LAK cell preserving conserving liquid in embodiment 1 than the LAK cell killing activity be kept in comparative example 1-2 respectively high by 22.3%, 14.5%, imitating target than when being 40:1, then improve 36.8%, 26.5% respectively.Use the LAK cell that the LAK cell-preservation liquid in embodiment 1 is preserved, under 4 DEG C of conditions, cell viability is more than 89%, and the killing activity of LAK cell does not have marked change, and the LAK cytoactive using cryopreserving liquid of the present invention frozen preserves LAK cell apparently higher than prior art cryopreserving liquid.
effect example 3, LAK cell surface marker expression
To using the LAK cell that in embodiment 1, comparative example 1 and comparative example 2, conserving liquid is preserved, preserving after 72 hours at 4 DEG C, carrying out the detection of surface marker.Concrete grammar is as follows: get 1 × 10
6individual LAK cell; 250g is centrifugal, and 5min removes supernatant; 2 times are cleaned by the PBS solution containing 10%FBS; Lucifuge adds CD3, CD56 antibody 2.5 μ L incubated at room 30min; 2 times are cleaned by the PBS solution containing 10%FBS; Filter with 500mLRPMI1640 medium re-suspended cell; Flow cytometer detects.
Flow cytometer detection result as shown in Figure 2.After knowing preservation 72h by Fig. 2, cell (effector cell) ratio that the CD3-CD56+ of the LAK cell in embodiment 1 expresses is 22.3%, the cell proportion that in comparative example 1, the CD3-CD56+ of LAK cell expresses is 12.1%, the cell proportion that in comparative example 2, the CD3-CD56+ of LAK cell expresses is 16.5%, illustrate that the surface marker expression rate of LAK cell in conserving liquid of the present invention is best, improve 84.3%, 35.2% respectively than comparative example 1-2.
LAK cell-preservation liquid of the present invention does not affect the expression of LAK cell surface marker; Use the LAK cell that LAK cell-preservation liquid of the present invention is preserved, under 4 DEG C of conditions, when cell viability is more than 89%, the expression of LAK cell surface marker is best.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a LAK cell-preservation liquid, is characterized in that, comprises human serum albumins, liquaemin, GL-B and preservation basal liquid, and described preservation basal liquid is compound electrolyte solution.
2. LAK cell-preservation liquid according to claim 1, is characterized in that, is made up of the following component of following quality percentage by volume:
Human serum albumins 1-5%,
Heparin sodium aqua 1-10%,
GL-B 1-5%,
Compound electrolyte solution surplus.
3. LAK cell-preservation liquid according to claim 2, is characterized in that, is made up of the following component of following quality percentage by volume:
Human serum albumins 1-2%,
Heparin sodium aqua 3-8%,
GL-B 2-3%,
Compound electrolyte solution surplus.
4. LAK cell-preservation liquid according to claim 3, is characterized in that, is made up of the following component of following quality percentage by volume:
Human serum albumins 1%,
Heparin sodium aqua 5%,
GL-B 2%,
Compound electrolyte solution surplus.
5. LAK cell-preservation liquid according to claim 1, it is characterized in that, described compound electrolyte solution is made up of the following component of following quality percentage by volume: sodium chloride 5.26%, gluconic acid sodium salt 5.02%, sodium acetate 3.68%, potassium chloride 0.37%, magnesium chloride 0.30%, water surplus.
6. the preparation method of the LAK cell-preservation liquid described in any one of claim 1-5, it is characterized in that, with a small amount of compound electrolyte solubilize liquaemin and GL-B, then add human serum albumins wherein, after supplying compound electrolyte solution, mix and obtain LAK cell-preservation liquid of the present invention.
7. a store method for LAK cell, is characterized in that, uses the LAK cell-preservation liquid described in any one of claim 1-5 that LAK cell is made cell preparation, preserves in the following temperature of normal temperature.
8. the store method of LAK cell according to claim 7, is characterized in that, the concentration of described cell preparation is 1 × 10
7/ mL.
9. the store method of LAK cell according to claim 7, is characterized in that, preservation condition is within normal temperature is preserved 12 hours.
10. the store method of LAK cell according to claim 7, is characterized in that, preservation condition is 4 DEG C and preserves within 72 hours.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107347871A (en) * | 2017-06-07 | 2017-11-17 | 江苏善之源健康科技有限公司 | A kind of immunocyte, which is fed back, preserves liquid |
| CN113615681A (en) * | 2021-08-27 | 2021-11-09 | 郑州源创吉因实业有限公司 | Frozen stock solution and frozen stock method for immune cells |
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