CN104480069A - Method of carrying out isolated culture on immune cells by virtue of peripheral blood - Google Patents
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- CN104480069A CN104480069A CN201410718862.8A CN201410718862A CN104480069A CN 104480069 A CN104480069 A CN 104480069A CN 201410718862 A CN201410718862 A CN 201410718862A CN 104480069 A CN104480069 A CN 104480069A
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- 210000005259 peripheral blood Anatomy 0.000 title claims abstract description 18
- 239000011886 peripheral blood Substances 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 12
- 210000002865 immune cell Anatomy 0.000 title abstract description 6
- 239000012679 serum free medium Substances 0.000 claims abstract description 28
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 21
- 108010002350 Interleukin-2 Proteins 0.000 claims abstract description 11
- 229920001917 Ficoll Polymers 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 5
- 210000002381 plasma Anatomy 0.000 claims description 50
- 210000004027 cell Anatomy 0.000 claims description 42
- 238000005119 centrifugation Methods 0.000 claims description 10
- 210000003677 hemocyte Anatomy 0.000 claims description 8
- 229940000351 hemocyte Drugs 0.000 claims description 8
- 239000012267 brine Substances 0.000 claims description 5
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 238000012258 culturing Methods 0.000 abstract 1
- 230000007812 deficiency Effects 0.000 abstract 1
- 239000004615 ingredient Substances 0.000 abstract 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 14
- 239000010410 layer Substances 0.000 description 9
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- 230000012010 growth Effects 0.000 description 6
- 102000004506 Blood Proteins Human genes 0.000 description 5
- 108010017384 Blood Proteins Proteins 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
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- 238000001514 detection method Methods 0.000 description 4
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- 238000002659 cell therapy Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
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- 230000003053 immunization Effects 0.000 description 2
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a method of carrying out isolated culture on immune cells by virtue of peripheral blood. The method comprises the following steps: centrifuging the peripheral blood for 10 min under a condition of 2000rpm and 4 DEG C, and collecting the upper-layer plasma to prepare a serum-free medium containing the plasma; adding Ficoll separation liquid and centrifuging; sucking a buffy coat, and washing to obtain PBMC; adding the serum-free medium in the PBMC, inoculating in the serum-free medium, and adding 20-300U/mL of IL-2 and 5-50ng/mL of IL-15; collecting the immune cells while culturing to the 13th to the 15th day. According to the method disclosed by the invention, the centrifuging time, the centrifuging rotational speed and the centrifuging temperature are adjusted, lots of the plasma can be obtained, and the stability of protein content in the plasma and no deficiencies of active factor ingredients can be ensured.
Description
Technical field
The present invention relates to a kind of cultural method of immunocyte, be specifically related to a kind of method utilizing peripheral blood separation and Culture immunocyte.
Background technology
Cellular immunotherapy therapy gathers human autoimmune's cell, through vitro culture, its quantity thousandfold is increased, targeting killing ability strengthens, and then feed back to human body to kill blood and tissue in pathogenic agent, cancer cells, sudden change cell, break immune tolerates, the immunological competence of activation and enhancing body, takes into account the double effects for the treatment of and health care.Cellular immunotherapy therapy main at present comprises cytokine induced kill cell (CIK) therapy, dendritic cell (DC) therapy, DC-CIK cell therapy, natural killer cell (NK) therapy, DC-T cell therapy etc.
Current immunocyte is cultivated mainly through serum free medium, but the serum free medium that regulation uses clinically needs to add the effect that certain density autologous plasma just can reach good amplification cultivation.Blood plasma is the important component part of blood, and in human body, the maximum level of autologous plasma is 55% of blood volume, therefore the autologous plasma obtaining maximum is as much as possible the key that current immune cell expansion is cultivated.
The centrifugation time of prior art is short or the too low plasma volume causing peripheral blood separating plasma to obtain of centrifugal force is few, and the long or too high meeting of centrifugal force of centrifugation time makes lower floor's hemocyte motility rate low, thus the immunocyte amount causing lower floor's PBMC inducing culture to obtain is few.In addition, blood plasma is few, and the immune cell media of equal autologous plasma concentration is less, and the final cell total amount obtained of cultivating is less.The invention provides a kind of new people's autologous plasma preparation method.Centrifugal rotating speed is 2000rpm, the time is 10min, temperature 4 DEG C, collects the motility rate that blood plasma does not affect lower floor's hemocyte under this condition, and can obtain more immunocyte total amount in immunocyte is cultivated.
In routine immunization cell cultures, except acquisition upper plasma, lower confluent monolayer cells need obtain peripheral blood mononuclear cell (PBMC) after being separated, then obtains the immunocytes such as NK, NKT, CIK through induction, amplification.Therefore the present invention is by optimizing the collection condition of blood plasma, can obtain more blood plasma, and ensure albuminous content and the active factor of blood plasma, lower floor PBMC obtains more immunocyte (NK/NKT/CIK etc.) through inducing culture.
The present invention is by optimizing centrifugal speed, centrifuging temperature and centrifugation time, find the centrifugal condition of best plasma collection, obtain larger plasma volume, ensure Cell viability higher after being separated simultaneously yet, and the content of active factor in plasma proteins and blood plasma does not all reduce.
The object of the invention is achieved through the following technical solutions:
Utilize a method for the immunocyte of peripheral blood separation and Culture, comprise the following steps:
(1) by peripheral blood centrifugal 10min under 2000rpm, 4 DEG C of conditions, collect upper plasma, be mixed with the serum free medium containing blood plasma, wherein the mass percent of blood plasma is 5%;
(2), after lower floor's hemocyte and physiological saline being mixed according to volume ratio 1:2 ~ 2:1, add Ficoll parting liquid, the volume ratio of mixed solution and Ficoll parting liquid is 1:2 ~ 2:1, and centrifugal force is 700g, centrifugal 20 ~ 40min;
(3) draw tunica albuginea layer, washing, namely obtains PBMC;
(4) in PBMC, add serum free medium, make cell density be 5 × 10
5~ 20 × 10
5/ ml, re-suspended cell precipitates, and is inoculated in serum free medium, and adds 20 ~ 300U/mL IL-2 and 5 ~ 50ng/mL IL-15;
(5) when cell density is greater than 2 × 10
6during/ml, add serum free medium, make cell density remain 5-10
5/ ml ~ 1 × 10
6/ ml, adds 20 ~ 300U/mL IL-2 and 5 ~ 50ng/mL IL-15; Cultivate 13rd ~ 15 days, collect immunocyte.
Brine twice is used in step (3) described washing.
The second layer that the described tunica albuginea layer of step (3) supernatant liquor after centrifugation counts from top to bottom.
The advantage that the present invention has relative to prior art and beneficial effect.
1., in existing peripheral blood separating plasma scheme, the centrifugal speed separated plasma of 3000rpm under employing normal temperature, while a large amount of blood plasma of acquisition, lower confluent monolayer cells has impaired greatly, affects the cultivation of follow-up immunization cell.The present invention have adjusted centrifugation time, centrifugal rotational speed and centrifuging temperature, acquisition blood plasma that can not only be relatively large, and can ensure not lacking of the stable and active factor composition of protein content in blood plasma;
2. in immunocyte culture scheme, part adopts the serum free medium containing foetal calf serum to cultivate at present, and foetal calf serum is cultivated and belonged to animal serum, may cause the pollution of animal derived pathogenic agent.So the present invention is by optimizing separating plasma condition, and the highly purified autologous plasma obtaining q.s, for cultivating immunocyte, has evaded the risk using animal serum to bring.
Summary of the invention
Accompanying drawing explanation
Fig. 1 is the aspect graph of NKT cell 100;
Fig. 2 is the flow cytometer detection of NKT cell;
Fig. 3 is the growth curve of NKT cell.
Embodiment
People's autologous plasma preparation method provided by the invention is as follows:
(1) medical anticoagulant tube blood sample collection, 8,5ml/ props up, altogether 40ml.
(2) 40mL peripheral blood is placed in 2-8 DEG C of constant temperature transport case, delivers to cell centre in 30min, cell centre receives peripheral blood, has registered relevant information
Peripheral blood in anticoagulant tube is transferred in 15mL centrifuge tube, divide 4 groups, often organize 10ml peripheral blood, carry out the separating plasma under different centrifugal condition respectively, as following table 1, and detecting plasma protein concentration (enzyme linked immunological kit), later separation PBMC amount and Cell viability, result is as following table 2, table 3 and table 4.
Separating plasma under the different centrifugal condition of table 1
Separated plasma under the condition of the different centrifugal rotational speed of table 2
As known from Table 2:
A), under different centrifugal speed condition, be separated the PBMC obtained and measure gap not quite, within the scope of reasonable error.
B) centrifugal speed increases, and plasma protein concentration reduces, and Cell viability reduces, and the Plasma volumes of acquisition increases, but rotating speed 2000rpm and above time, plasma content increases not obvious, has substantially reached the maximum of autologous plasma.
C) plasma protein concentration and Cell viability are obviously reducing higher than during 2000rpm, not obvious lower than 2000rpm difference.
Separated plasma under the condition of the different centrifuging temperature of table 3
As known from Table 3:
A), under different centrifuging temperature condition, it is little that the PBMC that Plasma volumes, plasma protein concentration (normal range 38-54g/L), separation obtain measures gap, within the scope of reasonable error;
B) centrifuging temperature is higher than 4 DEG C, and Cell viability obviously reduces; Lower than 4 DEG C, it is not obvious that Cell viability reduces trend.
Separated plasma under the condition of the different centrifugation time of table 4
As known from Table 4:
A) under different centrifugation time condition: be separated the PBMC obtained and measure difference in the reasonable scope;
B) centrifugation time is higher than 10min, and Cell viability obviously reduces; Centrifugation time is lower than 10min, and the Plasma volumes obtained obviously reduces.
More than test proof: 2000rpm, 4 DEG C, the separating plasma condition of 10min, can obtain a large amount of autologous plasmas, also can ensure the motility rate of the PBMC that density of plasma albumin is separated with lower floor.
The present invention, by after obtaining autologous plasma by the method for optimum, is separated lower floor's hemocyte, obtains corresponding PBMC.The autologous plasma obtained in the present invention is mixed with the serum free medium (LONZAX-VIVO 15 serum free medium) containing 5% autologous plasma concentration simultaneously, for cultivating PBMC, induced into NKT cell, and increase further, to meet the NKT cell concentration of clinical required cellular immunotherapy.
Embodiment 1:
1, by 20ml peripheral blood, centrifugal 10min under 2000rpm, 4 DEG C of conditions, collects autologous plasma 22ml, is mixed with the serum free medium containing 5% quality autologous plasma.
2, lower floor's hemocyte physiological saline 1:1 is diluted, add Ficoll parting liquid according to the ratio of 2:1,700
gcentrifugal 39min.
3, draw the second layer (from top to bottom) tunica albuginea layer in another clean centrifuge tube, with brine twice, be PBMC.
4, add a certain amount of serum free medium, make cell density be 1 × 10
6/ ml, re-suspended cell precipitates, and is inoculated in the serum free medium in T75 Tissue Culture Flask, and adds 20U/mL IL-2 and 5ng/mL IL-15.
5, after this, within every 3 days, get 20ul cell suspension to count, when cell density is greater than 2 × 10
6during/ml, add serum free medium, make cell density be 1 × 10
6/ ml, full dose adds 20U/mL IL-2 and 5ng/mL IL-15.
6, cultivate the 14th day, (Fig. 1 is the aspect graph of NKT cell 100 to collect NKT cell, left figure is amplification 100 times, right figure is amplification 400 times), carry out cell-surface antigens CD3, CD56 and detect (Fig. 2 is the flow cytometer detection of NKT cell), and the detection of NKT cell killing activity (result is as following table 5).Draw growth curve chart (Fig. 3 is the growth curve of NKT cell) according to cell counts simultaneously.
Fig. 1 observes at the inverted microscope of 100 times and 400 times, and cell state is good, and refractivity is strong, and agglomerating successful, meets immune cell growth feature.
Fig. 2 flow cytometer detection result shows, and PBMC is to NKT after differentiation-inducing 12 days, and CD3+CD56+ cell is 39%, and display inducing effect is good.
Fig. 3 shows, and cell, 1-4 days poor growths, belongs to inductive phase, and in logarithmic growth after 4 days, rate of propagation meets NKT cell normal growth state.
Table 5 and 6 illustrates: cultivate after 12 days, and NKT cell is to the killing activity of K562 cell up to 45.36%, and successful, illustrates that the NLT cell after cultivating has certain killing activity.
The killing activity of table 5NKT cell detects
| Cell type | Cell density | Motility rate |
| K562 (target cell) | 8.36×10 5cell/ml | 92.10％ |
| NKT (effector cell) | 1.53×10 6cell/ml | 95.30％ |
Table 6
| K562：NKT | 1:40 | 1:20 | 1:10 |
| Kill rate | 45.36％ | 43.52％ | 38.31％ |
Embodiment 2:
1, by 20ml peripheral blood, centrifugal 10min under 2000rpm, 4 DEG C of conditions, collects autologous plasma 22ml, is mixed with the serum free medium containing 5% quality autologous plasma.
2, lower floor's hemocyte physiological saline 1:2 is diluted, add Ficoll parting liquid according to the ratio of 1:2, the centrifugal 40min of 700g.
3, draw the second layer (from top to bottom) tunica albuginea layer in another clean centrifuge tube, with brine twice, be PBMC.
4, add a certain amount of serum free medium, make cell density be 5 × 10
5/ ml, re-suspended cell precipitates, and is inoculated in the serum free medium in T75 Tissue Culture Flask, and adds 300U/mL IL-2 and 50ng/mL IL-15.
5, after this, within every 3 days, get 20ul cell suspension to count, when cell density is greater than 2 × 10
6during/ml, add serum free medium, make cell density be 5 × 105/ml, full dose adds 300U/mL IL-2 and 50ng/mL IL-15.
6, cultivate the 14th day, collect NKT cell.
Embodiment 3:
1, by 20ml peripheral blood, centrifugal 10min under 2000rpm, 4 DEG C of conditions, collects autologous plasma 22ml, is mixed with the serum free medium containing 5% quality autologous plasma.
2, lower floor's hemocyte physiological saline 2:1 is diluted, add Ficoll parting liquid according to the ratio of 1:1, the centrifugal 20min of 700g.
3, draw the second layer (from top to bottom) tunica albuginea layer in another clean centrifuge tube, with brine twice, be PBMC.
4, add a certain amount of serum free medium, make cell density be 20 × 10
5/ ml, re-suspended cell precipitates, and is inoculated in the serum free medium in T75 Tissue Culture Flask, and adds 100U/mL IL-2 and 30ng/mL IL-15.
5, after this, within every 3 days, get 20ul cell suspension to count, when cell density is greater than 2 × 10
6during/ml, add serum free medium, make cell density be 20 × 10
5/ ml, full dose adds 100U/mL IL-2 and 30ng/mL IL-15.
6, cultivate the 14th day, collect NKT cell.
Claims (3)
1. utilize a method for peripheral blood separation and Culture immunocyte, it is characterized in that, comprise the following steps:
(1) by peripheral blood centrifugal 10min under 2000rpm, 4 DEG C of conditions, collect upper plasma, be mixed with the serum free medium containing blood plasma, wherein the mass percent of blood plasma is 5%;
(2), after lower floor's hemocyte and physiological saline being mixed according to volume ratio 1:2 ~ 2:1, add Ficoll parting liquid, the volume ratio of mixed solution and Ficoll parting liquid is 1:2 ~ 2:1, and centrifugal force is 700g, centrifugal 20 ~ 40min;
(3) draw tunica albuginea layer, washing, namely obtains PBMC;
(4) in PBMC, add serum free medium, make cell density be 5 × 10
5~ 20 × 10
5/ ml, re-suspended cell precipitates, and is inoculated in serum free medium, and adds 20 ~ 300U/mL IL-2 and 5 ~ 50ng/mL IL-15;
(5) when cell density is greater than 2 × 10
6during/ml, add serum free medium, make cell density remain 5-10
5/ ml ~ 1 × 10
6/ ml, adds 20 ~ 300U/mL IL-2 and 5 ~ 50ng/mL IL-15; Cultivate 13rd ~ 15 days, collect immunocyte.
2. method according to claim 1, is characterized in that, brine twice is used in step (3) described washing.
3. method according to claim 1, is characterized in that, the second layer counted from top to bottom of the described tunica albuginea layer of step (3) supernatant liquor after centrifugation.
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Cited By (6)
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| CN104911148A (en) * | 2015-07-14 | 2015-09-16 | 奥思达干细胞有限公司 | Human immunocompetent cell DC-CIK cytomedicine and effective preparation method thereof |
| CN105296425A (en) * | 2015-12-07 | 2016-02-03 | 黑龙江天晴干细胞股份有限公司 | Multi-cell immune preparation for treating tumors and preparation method of multi-cell immune preparation |
| CN106511975A (en) * | 2016-11-30 | 2017-03-22 | 广州赛莱拉干细胞科技股份有限公司 | Stem cell preparation, preparation method and application thereof |
| CN108567719A (en) * | 2017-09-26 | 2018-09-25 | 上海蕙禾生物科技事务所 | A kind of leukocyte extract and the preparation method and application thereof |
| CN110857435A (en) * | 2018-08-24 | 2020-03-03 | 上海中溢精准医疗科技有限公司 | Culture medium for culturing immune cells separated from cord blood and culture method thereof |
| CN114191373A (en) * | 2021-12-17 | 2022-03-18 | 内蒙古奕宏医学研究有限公司 | Method for quickly and simply extracting leukocyte extract from umbilical cord blood |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911148A (en) * | 2015-07-14 | 2015-09-16 | 奥思达干细胞有限公司 | Human immunocompetent cell DC-CIK cytomedicine and effective preparation method thereof |
| CN105296425A (en) * | 2015-12-07 | 2016-02-03 | 黑龙江天晴干细胞股份有限公司 | Multi-cell immune preparation for treating tumors and preparation method of multi-cell immune preparation |
| CN106511975A (en) * | 2016-11-30 | 2017-03-22 | 广州赛莱拉干细胞科技股份有限公司 | Stem cell preparation, preparation method and application thereof |
| CN108567719A (en) * | 2017-09-26 | 2018-09-25 | 上海蕙禾生物科技事务所 | A kind of leukocyte extract and the preparation method and application thereof |
| CN110857435A (en) * | 2018-08-24 | 2020-03-03 | 上海中溢精准医疗科技有限公司 | Culture medium for culturing immune cells separated from cord blood and culture method thereof |
| CN110857435B (en) * | 2018-08-24 | 2021-09-28 | 上海中溢精准医疗科技有限公司 | Culture medium for culturing immune cells separated from cord blood and culture method thereof |
| CN114191373A (en) * | 2021-12-17 | 2022-03-18 | 内蒙古奕宏医学研究有限公司 | Method for quickly and simply extracting leukocyte extract from umbilical cord blood |
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