CN102758259B - Method for constructing human peripheral blood immune cell bank - Google Patents
Method for constructing human peripheral blood immune cell bank Download PDFInfo
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Abstract
The invention discloses a method for constructing a human peripheral blood immune cell bank. The method comprises the following steps of: collecting human peripheral blood, separating autologous plasma, separating a peripheral blood mononuclear cell, separating a mononuclear cell by the peripheral blood mononuclear cell and freezing, separating a T lymphocyte by the peripheral blood mononuclear cell and freezing, separating a B lymphocyte by the peripheral blood mononuclear cell and freezing, separating an NK cell by the peripheral blood mononuclear cell and freezing, and encoding and putting in storage. According to the invention, immune cells of health or young people are separated and are respectively independently frozen, and relative numbers are stored and put into storage, so that the stored human immune cells have the characteristics of high activity, high purity and convenience for use, and fetal calf serum can be replaced by the human autologous plasma, so that introduction of a foreign protein can be avoided. Meanwhile, the method, provided by the invention, has the advantages of low cost, low requirements on laboratory conditions, and wide application.
Description
Technical field
The present invention relates to a kind of construction process of cell bank, relate in particular to the construction process in a kind of human peripheral immunocyte storehouse.
Background technology
Immunocyte refers to all cells relevant with immunne response, mainly comprises T cell, B cell, killer cell, natural killer cell, monocyte etc.In recent years tumour and virus the immunotherapy technology slowly be applied to clinical.As tumor vaccine and adoptive immunotherapy, these methods mainly are to feed back to improve patient's immunizing power by the immunocyte from body then through stimulated in vitro, cultivation, amplification, and such cell mainly contains DC at present, CIK, NK, DC-CIK etc.
These immunocyte treatments all mainly are the peripheral bloods of taking from patient oneself, realize through a series of operation then by obtaining peripheral blood mononuclear cell, but the colony that generally accepts the immunocyte treatment mainly is tumour or virus infection crowd, general this type of people's is older, and suffers from disease, the function of the immunocyte of itself is very low, and this moment, isolated activity of immune cells was very undesirable.So, when immunological competence at an early age is strong, isolate immunocyte and preserve and get up to have very large meaning.
For the ease of the effective storage of people and using-system and cell resource, worldwide there have been various kinds of cell storehouse or tissue bank to set up at present, such as umbilical hemopoietic stem cell storehouse, cell banks such as mesenchyma stem cell.So-called cell bank is to be about the place that stores cell and collect related data in-196 ℃ of liquid nitrogen.Yet, still unmanned autologous peripheral blood immunocyte storehouse foundation before key makes eye bright, therefore, make up the people and become peripheral body immunocyte storehouse, the immunocyte people is healthy or at an early age is built the storehouse by certain method, be fully to preserve and utilize existing immunocyte resources effective method, it provides strong support for storage person when needs adopt the immunocyte treatment technology, significant.
At present, frozen human peripheral immunocyte, normally direct frozen human peripheral blood single nucleus cell.But, the human peripheral blood single nucleus cell complicated component, existing immunocyte has non-immunocyte again, even immunocyte also is the combination of broad variety cell, these cells are had nothing in common with each other to frozen conditional request, if it is they are directly frozen together, motility rate is very not undesirable when not being the suitableeest, frozen mononuclearcell recovery because of frozen condition, can not satisfy clinical requirement.Simultaneously, at present the frozen storing liquid of freeze-stored cell generally all contains foetal calf serum, and foetal calf serum contains multiple protein, and compares with human body and to belong to foreign protein, has hidden danger in the application.Also have report to use the serum-free frozen storing liquid, but the serum-free frozen storing liquid is expensive, and complicated component, be difficult to clinical expansion.
Summary of the invention
At the deficiencies in the prior art, the purpose of this invention is to provide the construction process in a kind of human peripheral immunocyte storehouse.
The construction process in human peripheral immunocyte of the present invention storehouse, step comprises: gather human peripheral, separate autologous plasma, separate peripheral blood mononuclear cell, by peripheral blood mononuclear cell separating monocytic cell and frozen, separate T lymphocyte and frozen by peripheral blood mononuclear cell, separate bone-marrow-derived lymphocyte and frozen by peripheral blood mononuclear cell, separate NK cell and frozen by peripheral blood mononuclear cell, the coding warehouse-in; It is characterized in that:
Before described human peripheral was gathered, donor should be had a medical check-up and be met the standard of donating blood;
The method of described separation autologous plasma is: the peripheral blood of gathering is forwarded in the centrifuge tube, with 1000-2500 rev/min centrifugal 5～15 minutes, get supernatant liquor and obtain autologous plasma, 56 ℃ of deactivations are after 30 minutes, put-20 ℃ frozen, standby;
The method of described separation peripheral blood mononuclear cell is: the throw out behind the above-mentioned removal autologous plasma is by volume measured meter dilute 1 times with physiological saline or PBS, the hydroxyethyl starch solution that adds the 1wt%～6wt% of its 0.5～2 times of volume again, left standstill behind the mixing 10～40 minutes, isolate supernatant, and supernatant liquor is joined on the lymphocyte separation medium liquid level of equal volume amounts, 2000～5000 rev/mins centrifugal 10～30 minutes, divide three layers in the pipe of centrifugal back, get intermediate layer cell and namely obtain peripheral blood mononuclear cell;
Described separating monocytic cell and frozen method are: with the peripheral blood mononuclear cell that obtains according to 0.1～10 * 10
7The density of/ml is suspended in the substratum that component is 10 wt%FBS+90 wt%1640, and is divided in the 150cm of sterilization according to the amount of 30ml/ bottle
2In the Tissue Culture Flask, leave standstill 1h～5h, draw supernatant liquor then, standby; The residue attached cell is monocyte, with physiological saline or PBS adherent monocyte is blown and beaten, and 800～1500 rev/mins after centrifugal 5～10 minutes, with 1～10 * 10
5Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10 wt%DMSO+6wt% hydroxyethylamyles+84 wt% autologous plasmas;
The lymphocytic method of described separation T is: the supernatant liquor that adopts immunomagnetic beads method that separating monocytic cell is collected carries out the sorting of T lymphocyte, immunomagnetic beads method adopts positive separating method, antibody is selected CD3 antibody for use, isolate behind the T lymphocyte 800～1500 rev/mins centrifugal 5～10 minutes, again with 0.1～10 * 10
7Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10 wt%DMSO+6 wt% methylcellulose gum 400CP+84 wt% autologous plasmas;
The method of described separation bone-marrow-derived lymphocyte is: continue to carry out the bone-marrow-derived lymphocyte sorting with separating the supernatant liquor of the lymphocytic method of T to the separating monocytic cell collection, the positive separating method of same employing, antibody adopts CD19 or CD20, isolate behind the bone-marrow-derived lymphocyte 800～1500 rev/mins centrifugal 5～10 minutes, again with 0.1～10 * 10
7Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10 wt%DMSO+6 wt% Dextran 40s+84 wt% autologous plasmas;
The method of described separation NK cell is: after immunomagnetic beads method was separated T and bone-marrow-derived lymphocyte, remaining cell was the NK cell, behind centrifugal 5～10 minutes of 800～1500 rev/mins in described cell, with 0.1～10 * 10
6Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10 wt%DMSO+6 wt% trehaloses+84 wt% autologous plasmas;
The method of described coding warehouse-in is: number according to everyone group system separating also frozen various cells, and personal information is comprised numbering and preserves the unified computer of importing of positional information, for management and retrieval.
In the construction process in above-mentioned human peripheral immunocyte storehouse: the method for described separation peripheral blood mononuclear cell preferably: will remove throw out behind the autologous plasma and by volume measure meter with 1 times of physiological saline or PBS dilution, the hydroxyethyl starch solution that adds the 4wt% of its 0.5 times of volume again, left standstill behind the mixing 30 minutes, isolate supernatant, and supernatant liquor is joined on the lymphocyte separation medium liquid level of equal volume amounts, 3000 rev/mins centrifugal 20 minutes, divide three layers in the pipe of centrifugal back, get intermediate layer cell and namely obtain peripheral blood mononuclear cell.
The construction process in human peripheral immunocyte provided by the invention storehouse is isolated monocyte, T lymphocyte, bone-marrow-derived lymphocyte and NK sample T cell to health or young adult's immunocyte, and it is frozen separately respectively, association number is preserved warehouse-in, make people's immunocyte of preservation have active high, purity characteristics high, easy to use, and when preparation people periphery mononuclearcell, the present invention isolates human plasma, the personnel selection autologous plasma can replace foetal calf serum to use, and has avoided the introducing foreign protein.
The present invention compared with prior art has the following advantages:
The immunocyte storehouse that utilizes the present invention to set up, immunocyte are in optimum activity constantly before frozen, when the depositary used the immunocyte of this storehouse preservation to carry out the autoimmune cell treatment, cytoactive was more eager to excel in whatever one does than the immunologic cellular activity that blood sampling at that time separates.
The immunocyte storehouse that utilizes the present invention to set up, what preserve in the storehouse is each immunocyte subgroup, convenient during use, the immunocyte of a certain subgroup of can directly recovering is induced, amplification.
The inventive method has that cost is low, laboratory condition is less demanding, has a extensive future, and provides a large amount of cell resource bases for clinical or theoretical investigation.
Description of drawings
The construction process schema in Fig. 1 human peripheral immunocyte of the present invention storehouse.
Embodiment
The construction process in embodiment 1 human peripheral immunocyte storehouse
(1), gather human peripheral: check UP to supplying, according to the standard of donating blood it is checked, conformance with standard person draws blood two days later;
(2), separate autologous plasma: the peripheral blood that extracts is transferred in the centrifuge tube, in whizzer with 1500 rev/mins centrifugal 10 minutes, get supernatant liquor, 56 ℃ of deactivations 30 minutes, it is ℃ frozen, standby to finish postposition-20.
(3), separate peripheral blood mononuclear cell: above-mentioned steps is removed throw out behind the autologous plasma by volume measure meter with 1 times of physiological saline or PBS dilution, the hydroxyethyl starch solution that adds the 4wt% of its 0.5 times of volume again, left standstill behind the mixing 30 minutes, isolate supernatant, and supernatant liquor is joined on the lymphocyte separation medium liquid level of equal volume amounts, 3000 rev/mins centrifugal 20 minutes, divide three layers in the centrifugal back pipe, get intermediate layer cell and namely obtain peripheral blood mononuclear cell.
(4), separating monocytic cell and frozen: the mononuclearcell that step 3 is obtained is according to 0.1-10 * 10
7The density of/ml is suspended in the substratum that is divided into 10wt%FBS+90wt%1640, and is divided in the 150cm of sterilization according to the amount of 30ml/ bottle
2In the Tissue Culture Flask, leave standstill 4h～5h, draw supernatant liquor then, standby; The residue attached cell is monocyte.With physiological saline or PBS adherent monocyte is blown and beaten gently, 1200 rev/mins centrifugal 10 minutes, the back with 1～10 * 10
5Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% hydroxyethylamyle+84wt% autologous plasma.
(5), separate the T lymphocyte: the employing immunomagnetic beads method sub-elects the T lymphocyte in the supernatant liquor of drawing in the step 4, immunomagnetic beads method adopts positive separating method, antibody is selected CD3 antibody for use, isolate behind the T lymphocyte 1200 rev/mins centrifugal 10 minutes, the back is with 0.1～10 * 10
7Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% methylcellulose gum 400CP+84wt% autologous plasma.
(6), separate bone-marrow-derived lymphocyte: continue to carry out the bone-marrow-derived lymphocyte sorting with separating the supernatant liquor of the lymphocytic method of T to the separating monocytic cell collection, the positive separating method of same employing, adopting antibody is CD19 or CD20, isolate behind the bone-marrow-derived lymphocyte 1200 rev/mins centrifugal 10 minutes, the back is with 0.1-10 * 10
7Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% Dextran 40+84wt% autologous plasma.
(7), separate the NK cell: after immunomagnetic beads method was separated T and bone-marrow-derived lymphocyte, remaining cell was the NK cell, behind centrifugal 10 minutes of 1200 rev/mins in described cell, with 0.1～10 * 10
6Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% trehalose+84wt% autologous plasma.
(8), coding warehouse-in: number according to everyone group system separating also frozen various cells, and personal information is comprised numbering and preserves the unified computer of importing of positional information, for management and retrieval.
The construction process in embodiment 2 human peripheral immunocyte storehouses
(1), gather human peripheral: check UP to supplying, according to the standard of donating blood it is checked, conformance with standard person draws blood two days later;
(2), separate autologous plasma: the peripheral blood that extracts is transferred in the centrifuge tube, in whizzer with 2500 rev/mins centrifugal 5 minutes, get supernatant liquor, 56 ℃ of deactivations 30 minutes, it is ℃ frozen, standby to finish postposition-20.
(3), separate peripheral blood mononuclear cell: above-mentioned steps is removed throw out behind the autologous plasma by volume measure meter with 1 times of physiological saline or PBS dilution, the hydroxyethyl starch solution that adds the 3wt% of its 1 times of volume again, left standstill behind the mixing 40 minutes, isolate supernatant, and supernatant liquor is joined on the lymphocyte separation medium liquid level of equal volume amounts, 4000 rev/mins centrifugal 15 minutes, divide three layers in the centrifugal back pipe, get intermediate layer cell and namely obtain peripheral blood mononuclear cell.
(4), separating monocytic cell and frozen: the mononuclearcell that step 3 is obtained is according to 0.1-10 * 10
7The density of/ml is suspended in the substratum that is divided into 10wt%FBS+90wt%1640, and is divided in the 150cm of sterilization according to the amount of 30ml/ bottle
2In the Tissue Culture Flask, leave standstill 5h, draw supernatant liquor then, standby; The residue attached cell is monocyte.With physiological saline or PBS adherent monocyte is blown and beaten gently, 1500 rev/mins centrifugal 5 minutes, the back with 1～10 * 10
5Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% hydroxyethylamyle+84wt% autologous plasma.
(5), separate the T lymphocyte: the employing immunomagnetic beads method sub-elects the T lymphocyte in the supernatant liquor of drawing in the step 4, immunomagnetic beads method adopts positive separating method, antibody is selected CD3 antibody for use, isolate behind the T lymphocyte 1500 rev/mins centrifugal 5 minutes, the back is with 0.1～10 * 10
7Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% methylcellulose gum 400CP+84wt% autologous plasma.
(6), separate bone-marrow-derived lymphocyte: continue to carry out the bone-marrow-derived lymphocyte sorting with separating the supernatant liquor of the lymphocytic method of T to the separating monocytic cell collection, the positive separating method of same employing, adopting antibody is CD19 or CD20, isolate behind the bone-marrow-derived lymphocyte 1500 rev/mins centrifugal 5 minutes, the back is with 0.1-10 * 10
7Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% Dextran 40+84wt% autologous plasma.
(7), separate the NK cell: after immunomagnetic beads method was separated T and bone-marrow-derived lymphocyte, remaining cell was the NK cell, behind centrifugal 5 minutes of 1500 rev/mins in described cell, with 0.1～10 * 10
6Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% trehalose+84wt% autologous plasma.
(8), coding warehouse-in: number according to everyone group system separating also frozen various cells, and personal information is comprised numbering and preserves the unified computer of importing of positional information, for management and retrieval.
Claims (2)
1. the construction process in a human peripheral immunocyte storehouse, step comprises: a blood that selection meets the standard of donating blood is human peripheral, separated plasma, separate peripheral blood mononuclear cell, by peripheral blood mononuclear cell separating monocytic cell and frozen, separate T lymphocyte and frozen by peripheral blood mononuclear cell, separate bone-marrow-derived lymphocyte and frozen by peripheral blood mononuclear cell, separate NK cell and frozen by peripheral blood mononuclear cell, the coding warehouse-in; It is characterized in that:
The method of described separated plasma is: with peripheral blood with 1000-2500 rev/min centrifugal 5～15 minutes, get supernatant liquor and obtain blood plasma, 56 ℃ of deactivations are after 30 minutes, put-20 ℃ frozen, standby;
The method of described separation peripheral blood mononuclear cell is: the throw out behind the above-mentioned removal blood plasma is by volume measured meter dilute 1 times with physiological saline or PBS, the hydroxyethyl starch solution that adds the 1wt%～6wt% of its 0.5～2 times of volume again, left standstill behind the mixing 10～40 minutes, isolate supernatant, and supernatant liquor is joined on the lymphocyte separation medium liquid level of equal volume amounts, 2000～5000 rev/mins centrifugal 10～30 minutes, divide three layers in the pipe of centrifugal back, get intermediate layer cell and namely obtain peripheral blood mononuclear cell;
Described separating monocytic cell and frozen method are: with the peripheral blood mononuclear cell that obtains according to 0.1～10 * 10
7The density of/ml is suspended in the substratum that component is 10wt%FBS+90wt%1640, and is divided in the 150cm of sterilization according to the amount of 30ml/ bottle
2In the Tissue Culture Flask, leave standstill 1h～5h, draw supernatant liquor then, standby; The residue attached cell is monocyte, with physiological saline or PBS adherent monocyte is blown and beaten, and 800～1500 rev/mins after centrifugal 5～10 minutes, with 1～10 * 10
5Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% hydroxyethylamyle+84wt% blood plasma;
The lymphocytic method of described separation T is: the supernatant liquor that adopts immunomagnetic beads method that separating monocytic cell is collected carries out the sorting of T lymphocyte, immunomagnetic beads method adopts positive separating method, antibody is selected CD3 antibody for use, isolate behind the T lymphocyte 800～1500 rev/mins centrifugal 5～10 minutes, again with 0.1～10 * 10
7Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% methylcellulose gum 400CP+84wt% blood plasma;
The method of described separation bone-marrow-derived lymphocyte is: continue to carry out the bone-marrow-derived lymphocyte sorting with separating the supernatant liquor of the lymphocytic method of T to the separating monocytic cell collection, the positive separating method of same employing, antibody adopts CD19 or CD20, isolate behind the bone-marrow-derived lymphocyte 800～1500 rev/mins centrifugal 5～10 minutes, again with 0.1～10 * 10
7Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% Dextran 40+84wt% blood plasma;
The method of described separation NK cell is: after immunomagnetic beads method was separated T and bone-marrow-derived lymphocyte, remaining cell was the NK cell, behind centrifugal 5～10 minutes of 800～1500 rev/mins in described cell, with 0.1～10 * 10
6Density carry out packing and liquid nitrogen cryopreservation, the frozen storing liquid prescription is 10wt%DMSO+6wt% trehalose+84wt% blood plasma;
The method of described coding warehouse-in is: separation and frozen various cells are numbered according to every part of group system, and cell information is comprised numbering and preserves the unified computer of importing of positional information, be used for management and retrieval.
2. the construction process in human peripheral immunocyte storehouse according to claim 1, it is characterized in that: the method for described separation peripheral blood mononuclear cell is: will remove throw out behind the blood plasma and by volume measure meter with 1 times of physiological saline or PBS dilution, the hydroxyethyl starch solution that adds the 4wt% of its 0.5 times of volume again, left standstill behind the mixing 30 minutes, isolate supernatant, and supernatant liquor is joined on the lymphocyte separation medium liquid level of equal volume amounts, 3000 rev/mins centrifugal 20 minutes, divide three layers in the pipe of centrifugal back, get intermediate layer cell and namely obtain peripheral blood mononuclear cell.
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Cited By (1)
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| CN105505868B (en) * | 2015-12-31 | 2019-02-15 | 北京弘润天源基因生物技术有限公司 | Separate the separation method of peripheral blood immunocyte |
| CN105543168B (en) * | 2015-12-31 | 2019-01-22 | 北京弘润天源基因生物技术有限公司 | The storage and transportation resources of immunocyte |
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| CN106085954A (en) * | 2016-06-24 | 2016-11-09 | 安徽未名细胞治疗有限公司 | A kind of a large amount of human peripheral PBMC fast separating process |
| CN106635984A (en) * | 2016-11-16 | 2017-05-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Human umbilical cord blood immune cell bank establishing method |
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| CN112029720A (en) * | 2020-08-24 | 2020-12-04 | 海南优尼科尔生物科技有限公司 | Construction method of human peripheral blood NK cell bank |
| CN114762497B (en) * | 2021-01-11 | 2023-08-11 | 京东方再生医学科技有限公司 | Cell cryopreservation liquid and cell cryopreservation method |
| CN113445132A (en) * | 2021-04-06 | 2021-09-28 | 海南优尼科尔生物科技有限公司 | Method for constructing CIK cell bank |
| CN112852729A (en) * | 2021-04-16 | 2021-05-28 | 天津市环湖医院 | Method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissues |
| CN113174369B (en) * | 2021-04-23 | 2022-10-18 | 北京起源爱心生物科技有限公司 | Establishment method of immune cell bank with pleiotropic immunoregulation function |
| CN113697146B (en) * | 2021-08-20 | 2023-03-24 | 苏州博腾生物制药有限公司 | Cell subpackaging method in cell bank building process |
| CN114807031A (en) * | 2022-05-13 | 2022-07-29 | 山东赛恩福干细胞工程集团有限公司 | Construction method of human peripheral blood immune cell bank and stem cell bank |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6140470A (en) * | 1995-06-30 | 2000-10-31 | Yale University | Human monoclonal anti-tumor antibodies |
| CN100344757C (en) * | 2005-10-18 | 2007-10-24 | 天津昂赛细胞基因工程有限公司 | Human placenta, umbilical cord mesenchyma stemcell stock and its construction method |
| CN101608174B (en) * | 2009-07-23 | 2012-02-22 | 章毅 | Construction method of human umbilical cord mesenchyma stem cell |
| CN102002475B (en) * | 2010-03-10 | 2011-12-21 | 和泽生物科技有限公司 | Method for obtaining fat adult stem cells of human and method for establishing stem cell library |
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2012
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105219709A (en) * | 2015-09-23 | 2016-01-06 | 上海吉泉生物技术有限公司 | Dan Caifa sets up immunocyte storehouse and application |
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