CN102758259A - Method for constructing human peripheral blood immune cell bank - Google Patents
- ️Wed Oct 31 2012
CN102758259A - 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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- CN102758259A CN102758259A CN2012102697374A CN201210269737A CN102758259A CN 102758259 A CN102758259 A CN 102758259A CN 2012102697374 A CN2012102697374 A CN 2012102697374A CN 201210269737 A CN201210269737 A CN 201210269737A CN 102758259 A CN102758259 A CN 102758259A Authority
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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 is meant all cells relevant with immunne response, mainly comprises T cell, B cell, killer cell, nk cell, monocyte etc.In recent years tumour with virus the immunotherapy technology slowly be applied to clinical.Like tumor vaccine and adoptive immunotherapy, these methods mainly are to feed back the immunizing power that improves the patient through 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 series of operation then through obtaining PMNC; But the colony that generally accepts the immunocyte treatment mainly is tumour or virus infection crowd, and generally 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 was set up before key made eye bright, and therefore, made up the people and became peripheral body immunocyte storehouse; The immunocyte people is healthy or at an early age is built the storehouse through 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 righttest, 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 cost an arm and a leg, and complicated component, be difficult to clinical expansion.
Summary of the invention
To the deficiency of 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 according to the invention storehouse, step comprises: gather human peripheral, separate autologous plasma; Separate PMNC; By PMNC separating monocytic cell and frozen, separate T lymphocyte and frozen by PMNC, separate bone-marrow-derived lymphocyte and frozen by PMNC; Separate NK cell and frozen by PMNC, the coding warehouse-in; It is characterized in that:
Before said human peripheral was gathered, donor should be had a medical check-up and met the standard of donating blood;
The method of said 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 and obtain autologous plasma, 56 ℃ of deactivations are after 30 minutes, put-20 ℃ frozen, subsequent use;
The method of said separation PMNC is: the throw out behind the above-mentioned removal autologous plasma is by volume measured meter dilute 1 times with saline water 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 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 promptly obtain PMNC;
Said separating monocytic cell and frozen method are: with the PMNC 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 then, subsequent use; The residue attached cell is monocyte, with saline water 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 said separation T is: the supernatant 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 said separation bone-marrow-derived lymphocyte is: continue to carry out the bone-marrow-derived lymphocyte sorting with separating the supernatant 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 said 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 said 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 said 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 computingmachine of importing of positional information, be used for managing and retrieval.
In the construction process in above-mentioned human peripheral immunocyte storehouse: the method for said separation PMNC preferably: will remove throw out behind the autologous plasma and by volume measure meter with 1 times of saline water 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 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 promptly obtain PMNC.
The construction process in human peripheral immunocyte provided by the invention storehouse is isolated monocyte, T lymphocyte, bone-marrow-derived lymphocyte and NK appearance 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 isolate 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 immunocyte that uses this storehouse to preserve the depositary carried out the autoimmune cell treatment, the cytoactive isolating immunologic cellular activity of taking a blood sample than at that time was eager to excel in whatever one does.
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 a large amount of cell resource bases is provided for clinical or theoretical investigation.
Description of drawings
The construction process schema in Fig. 1 human peripheral immunocyte according to the 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, 56 ℃ of deactivations 30 minutes, accomplish rearmounted-20 ℃ frozen, subsequent use.
(3), separate PMNC: above-mentioned steps is removed throw out behind the autologous plasma by volume measure meter with 1 times of saline water 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, isolated supernatant; And supernatant 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 promptly obtain PMNC.
(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 then, subsequent use; The residue attached cell is monocyte.With saline water 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: adopt immunomagnetic beads method to sub-elect the T lymphocyte in the supernatant 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 a 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 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 said 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 computingmachine of importing of positional information, be used for managing 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, 56 ℃ of deactivations 30 minutes, accomplish rearmounted-20 ℃ frozen, subsequent use.
(3), separate PMNC: above-mentioned steps is removed throw out behind the autologous plasma by volume measure meter with 1 times of saline water 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, isolated supernatant; And supernatant 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 promptly obtain PMNC.
(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 then, subsequent use; The residue attached cell is monocyte.With saline water 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: adopt immunomagnetic beads method to sub-elect the T lymphocyte in the supernatant 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 a 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 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 said 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 computingmachine of importing of positional information, be used for managing and retrieval.
Claims (2)
1. the construction process in a human peripheral immunocyte storehouse, step comprises: gather human peripheral, separate autologous plasma; Separate PMNC; By PMNC separating monocytic cell and frozen, separate T lymphocyte and frozen by PMNC, separate bone-marrow-derived lymphocyte and frozen by PMNC; Separate NK cell and frozen by PMNC, the coding warehouse-in; It is characterized in that:
Before said human peripheral was gathered, donor should be had a medical check-up and met the standard of donating blood;
The method of said 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 and obtain autologous plasma, 56 ℃ of deactivations are after 30 minutes, put-20 ℃ frozen, subsequent use;
The method of said separation PMNC is: the throw out behind the above-mentioned removal autologous plasma is by volume measured meter dilute 1 times with saline water 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 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 promptly obtain PMNC;
Said separating monocytic cell and frozen method are: with the PMNC 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 then, subsequent use; The residue attached cell is monocyte, with saline water 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 said separation T is: the supernatant 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 said separation bone-marrow-derived lymphocyte is: continue to carry out the bone-marrow-derived lymphocyte sorting with separating the supernatant 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 said 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 said 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 said 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 computingmachine of importing of positional information, be used for managing and retrieval.
2. the construction process in human peripheral immunocyte storehouse according to claim 1; It is characterized in that: the method for said separation PMNC is: will remove throw out behind the autologous plasma and by volume measure meter with 1 times of saline water 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, isolated supernatant; And supernatant 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 promptly obtain PMNC.
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