CN105296432B - A method of T lymphocyte is induced into multipotential stem cell - Google Patents
A method of T lymphocyte is induced into multipotential stem cell Download PDFInfo
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Abstract
The present invention relates to technical field of cell culture, in particular to a kind of method for inducing T lymphocyte at multipotential stem cell.This method includes：T lymphocyte will be transfected containing the sendai virus of stem cell transcription factor gene, obtains infection cell；Infection cell is mixed with feeder cells, first culture is carried out using feeder cells culture medium, then feeder cells culture medium is abandoned, second culture is carried out using embryonic stem cell medium, then third culture is carried out using conditioned medium and the mixed liquor of human embryonic stem cell medium, the 4th culture is finally carried out using human embryonic stem cell medium, finally obtains multipotential stem cell.The present invention selects sendai virus as transfection carrier, avoids the genome conformity of virus into the genome of donorcells, improves the safety of iPS cell；Select periphery blood T lymphocyte as the donorcells of iPS cell, donor cell sources are simple；The method of the present invention can successfully obtain multipotential stem cell, and the conversion ratio and purity of multipotential stem cell are higher.
Description
Technical field
It is the present invention relates to technical field of cell culture, in particular to a kind of to induce T lymphocyte at multipotential stem cell
Method.
Background technique
Embryonic stem cell (embryonic stem cell, ESCs, abbreviation ES, EK or ESC cell) is that body early embryo is (former
Before the gastrula phase) or original sexual gland in a kind of cell for separating, it has in vitro culture infinite multiplication, self-renewing and more
To the characteristic of differentiation.No matter in vitro or vivo environment, ES cell can be induced to differentiate into the almost all of cell of body
Type.Embryonic stem cell is in the great application value in the fields such as organizational project and regenerative medicine.
As the ES cell for being referred to as " seed cell ", lot of materials is provided for clinical histoorgan transplanting.People ES
For cell after immunological rejection gene knockout, reorientation induces end-organ to avoid interindividual graft rejection.In this way
The graft rejection problem between the allograft individual that annoying immune educational circles and medical field can be can solve always.ES cell is available
In cell therapy, cell therapy refers to the human body cell directly transplanting of genetically engineered mistake or input in patient body, reaches
Cure and control the purpose of disease.ES cell remains to steadily be proliferated passage in vitro after genetic manipulation.It is to carry with ES cell
Body, through in-vitro directed transformation, make gene integrates number, site, expression degree and the stability and screening operation of being inserted into gene
Deng all carrying out on a cellular level, be easy to get stablize, satisfied transgenic ES cells system, led to overcome in current gene therapy
The integration and expression for entering gene are difficult to control, and the cell as genetic manipulation is not easy steadily to be transfected and be proliferated in vitro
Passage opens new approach.
However, since it is difficult to obtain and be related to sensitive ethics problem, so that research was once falling into awkward condition.2006
Year August, Japanese Yamanaka research group import 4 kinds of transcription factor genes (Oct4, Sox2, c- into l cell
Myc and Klf4), it is successfully reprogrammed as ESCs sample inductive pluripotent stem cells
(inducedpluripotentstemcells, iPSCs).The end of the year 2007, Japanese Yamanaka group and U.S. Thomson are small
This revolutionary character technology is achieved by group in human cell in succession.The research of iPSCs, which is one, has initiative therapeutic
The basic research of clone avoids ESCs for a long time and is difficult to the problem of obtaining and easily causing dispute of ethic, cures for stem cell
It learns application to open up a new way, it is considered to be the great discovery of stem cell field or even entire field of biology.Including
Many scientists including Univ Edinburgh UK Ian professor Wilmut think that inductive pluripotent stem cells are only stem cell
Following research direction.IPSCs cell not only for stem-cell research have important theory significance, and cell therapy,
The great application value in the fields such as tissue and organ regeneration, drug screening and evaluation.
However, the safety of iPSCs is the problem of must be taken into consideration before clinical application.For mediating the majority disease of target gene
Poisonous carrier can be integrated into target cell genome, it is possible to activated oncogene.For the security risk for eliminating iPSCs, researcher
Seeking a kind of safer transcription factor lead-in mode always.
Summary of the invention
In view of this, the present invention provides a kind of by T lymphocyte induction into the method for multipotential stem cell.This method can be at
Function obtains multipotential stem cell, highly-safe, and conversion ratio and purity are higher.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of by T lymphocyte induction into the method for multipotential stem cell, includes the following steps：
T lymphocyte will be transfected containing the sendai virus of stem cell transcription factor gene, obtains infection cell；
Infection cell is mixed with feeder cells, carries out the first culture, carries out the using human embryonic stem cell medium
Two cultures, then carry out third training using the conditioned medium of feeder cells and the mixed liquor of human embryonic stem cell medium
It supports, the 4th culture is finally carried out using human embryonic stem cell medium, obtains multipotential stem cell.
First culture be specially：Infection cell is mixed with feeder cells, is trained using feeder cells culture medium
It supports, then abandons feeder cells culture medium.
The present invention is using sendai virus as transfection carrier, using periphery blood T lymphocyte as donorcells, first by infection cell with
It after feeder cells mixed culture, is cultivated using human embryonic stem cell medium, then uses conditioned medium and people's embryo
The mixed liquor of tire stem cell media is cultivated, and is finally cultivated, is successfully obtained more using human embryonic stem cell medium
Energy stem cell, the conversion ratio and purity of multipotential stem cell are higher.
In some embodiments provided by the invention, stem cell transcription factor is in OCT4, SOX2, C-MYC or KLF4
One or more kinds of mixtures.
Preferably, transfection is 24~36h of transfection under the conditions of 37 DEG C.
Preferably, it transfects to be transfected for 24 hours under the conditions of 37 DEG C.
In some embodiments provided by the invention, the formula of conditioned medium is：P2 for MEF cell (mice embryonic at
Fibrocyte) culture supernatant.
In some embodiments provided by the invention, embryonic stem cell medium is human embryonic stem cell medium.
In some embodiments provided by the invention, the formula of human embryonic stem cell medium is：DMEM/F12, serum replace
For object, β-thioglycol, 1% nonessential amino acid, FGF, Basic Fibroblast Growth Factor.
Preferably, the volume ratio of conditioned medium and embryonic stem cell medium is 1：(1~1.5).
Preferably, the volume ratio of conditioned medium and embryonic stem cell medium is 1：1.
Preferably, transfection use help and turn agent as polybrene.
Preferably, the time of the first culture is 1 day, the time of the second culture is 9~11 days.
Preferably, the time of the second culture is 9 days.
Preferably, the time of third culture is 8~10 days, the time of the 4th culture is 7~15 days.
Preferably, the time of third culture is 8 days, and the time of the 4th culture is 7 days.
The present invention provides a kind of by T lymphocyte induction into the method for multipotential stem cell.The method comprising the steps of：It will contain
There is the sendai virus transfection T lymphocyte of stem cell transcription factor gene, obtains infection cell；Infection cell is thin with feeder layer
Born of the same parents' mixing carries out the first culture, carries out the second culture using human embryonic stem cell medium, then uses conditioned medium and people
The mixed liquor of embryonic stem cell medium carries out third culture, finally carries out the 4th culture using human embryonic stem cell medium,
Obtain multipotential stem cell.The present invention at least has one of following advantage：
1, the present invention selects sendai virus as transfection carrier, avoids the genome conformity of virus to the gene of donorcells
In group, the safety of iPS cell is improved；
2, the present invention selects periphery blood T lymphocyte as the donorcells of iPS cell, and donor cell sources are simple；
3, the present invention is using sendai virus as transfection carrier, using periphery blood T lymphocyte as donorcells, first by infection cell
It after feeder cells mixed culture, is cultivated using human embryonic stem cell medium, then uses conditioned medium and people
The mixed liquor of embryonic stem cell medium is cultivated, and is finally cultivated, can successfully be obtained using human embryo stem cell culture solution
Multipotential stem cell, the conversion ratio and purity of multipotential stem cell are higher, for by iPS cell be applied to disease of immune system or
Certain theoretical basis is established in tumor vaccine cells treatment.
Detailed description of the invention
Fig. 1 shows the form of the iPS cell of acquisition；
Fig. 2 shows alkaline phosphatase staining result.
Specific embodiment
The invention discloses a kind of by T lymphocyte induction into the method for multipotential stem cell, and those skilled in the art can be with
Present disclosure is used for reference, realization of process parameters is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications are to ability
It is it will be apparent that they are considered as being included in the present invention for field technique personnel.Method and application of the invention has been led to
Preferred embodiment is crossed to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to this paper institute
The methods and applications stated are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Term is explained：
Induce multi-potent stem cell (induced pluripotent stem cells, iPS cells)：Pass through certain way
Channel genes related with cells pluripotency into differentiated body cell, or are added the small of some booster actions by diameter simultaneously
Molecular compound makes body cell dedifferente reprogramming to return to embryonic stem cell state, and cell obtained is iPS cell.
It is provided by the invention to induce T lymphocyte at agents useful for same, the city instrument Jun Keyou in the method for multipotential stem cell
Field is bought.
The formula of conditioned medium is：When P2 is for MEF cell culture, the culture supernatant of the culture MEF cell of collection, as
Conditioned medium；
The formula of human embryonic stem cell medium is：89% human embryo stem cell culture solution：knockoutTM DMEM/F12
(Gibco)+knockoutTMSerum replacement (KSR) (KnockOut serum substitute；Gibco)+β-thioglycol
+ 1%NEAA (nonessential amino acid) (Sigma)+10ng/mL basic FGF, recombinant human (basic fibroblast
Growth factor) (PeproTech)；
It helps and turns agent：polybrene.
Below with reference to embodiment, the present invention is further explained：
The preparation that embodiment 1 induces multi-potent stem cell
(1) separation and sorting of total T lymphocyte
1) peripheral blood in anticoagulant tube is transferred in centrifuge tube, 400-500g (centrifugal force) is centrifuged 5-10min, centrifugation knot
Lower layer's haemocyte is transferred in 50mL centrifuge tube after beam, the physiological saline that two volumes are added is diluted；
2) a new sepmate centrifuge tube (purchased from STEM CELL company) is taken, Ficoll lymphocyte separation medium is added
Haemocyte after dilution is transferred to sepmate centrifuge tube by (being purchased from STEM CELL company) 12mL, and 600-800g is centrifuged 15-
20min；
3) after being centrifuged, supernatant liquid is poured out, physiological saline is added and carries out washing 1 time；
4) CD3 is used+Sorting kit (being purchased from STEM CELL company) sorts T lymphocyte from above-mentioned mononuclearcell
Out.
(2) preparation of feeder cells
1) by the MEF cell recovery in P2 generation；
2) when MEF cell (mouse embryo fibroblast stem cell) fusion rate reaches 80~90%, MEF cell is passed
Generation.Then by 1-3 × 105Cell inoculation into six orifice plates；
3) processing 2 hours was carried out with the mitomycin C of 5-20 μ g/mL in 1st；
4) it changes MEF culture medium after 2 hours into, obtains feeder cells.
(3) culture of sendai virus transfection and iPS cell
1) according to sendai virus reprogramming kit operation：By the sendai virus containing OCT4, SOX2, C-MYC and KLF4
Solution is added in six orifice plates, and liquor capacity finally complements to 2mL, is eventually adding 2 helping for the μ of μ L5~20 g/mL and turns agent, by entire body
It after system mixes, is transferred in a hole of pre-coated good 6 orifice plates, is put into after shaking up in 37 DEG C of incubators, overnight.
2) cell that yesterday infects (the 1st day) after virus infection 24 hours, is taped against preprepared feeder cells
On.
3) it the 2nd day, changes DMEM high glucose medium into human embryonic cells culture solution, continues to cultivate.Change liquid one within every 2 days later
It is secondary.
4) it observes day by day, when cell long to the 5th~7 day, it is seen that small cell aggregation, cellular morphology obviously occur
Change, growth flocks together.
5) up to the 10th day, since MEF cell uses overlong time, therefore culture solution changes CM (conditioned into
Medium, conditioned medium) the normal culture solution of+hESC (human embryo stem cell) 1:1 (volume ratio) mixed liquor is thin to be supplied to
The nutrition of born of the same parents' abundance.
6) long to the 18th day to cell, clone can be chosen, the clone of embryonic stem cell sample is chosen to new MEF, is pressed
HESC (hESC) cultural method continues culture 7 days, amplification.IPS cell clone and normal human embryonic stem are thin
Born of the same parents are morphologically no different (Fig. 1) substantially.
The identification (alkaline phosphatase activities detection) that the cell of above-mentioned acquisition is carried out to iPS cell, according to alkaline phosphatase
Kit specification is operated, and specific operating procedure is as follows：
(1) cell culture fluid is sucked out and fixes 1~2min with 4% paraformaldehyde (PFA) with PBS solution rinse 2~3 times；
(2) fixer is sucked out, is washed 2 times with PBS solution；
(3) PBS solution is sucked out, it is primary with TBST solution rinse；
(4) prepare alkaline phosphatase reagent：[solution A (white)：Solution B (green)：Solution C=50ul：50ul：400ul]；
(5) plus enough staining reagents enable dyeing liquor to cover bottom hole, 10~15min of room temperature avoid light place enough；
(6) dyeing liquor is sucked out, it is primary with PBS buffer solution rinse, it is finally stored in PBS solution, microscopically observation dye
Color result (neoblast dyeing is indigo plant/purple, and the cell dyeing of differentiation is colourless).Blue cell is that differentiation is thin as the result is shown
Born of the same parents obtain multipotential stem cell.
The preparation of 2 multipotential stem cell of embodiment
(1) separation and sorting of total T lymphocyte
1) peripheral blood in anticoagulant tube is transferred in centrifuge tube, 400-500g (centrifugal force) is centrifuged 5-10min, centrifugation knot
Lower layer's haemocyte is transferred in 50mL centrifuge tube after beam, the physiological saline that two volumes are added is diluted；
2) a new sepmate centrifuge tube (purchased from STEM CELL company) is taken, Ficoll lymphocyte separation medium is added
Haemocyte after dilution is transferred to sepmate centrifuge tube by (being purchased from STEM CELL company) 12mL, and 600-800g is centrifuged 15-
20min；
3) after being centrifuged, supernatant liquid is poured out, physiological saline is added and carries out washing 1 time；
4) CD3 is used+Sorting kit (being purchased from STEM CELL company) sorts T lymphocyte from above-mentioned mononuclearcell
Out.
(2) preparation of feeder cells
1) the MEF cell in P2 generation (mouse embryo fibroblast stem cell) is recovered；
2) when MEF cell confluency reaches 80~90%, MEF cell is passed on.Then by 1-3 × 105It is thin
Born of the same parents are inoculated into six orifice plates；
3) processing 2 hours was carried out with the mitomycin C of 5-20 μ g/mL in 1st；
4) it changes MEF culture medium after 2 hours into, obtains feeder cells.
(3) culture of sendai virus transfection and iPS cell
1) according to sendai virus reprogramming kit operation：By the sendai virus containing OCT4, SOX2, C-MYC and KLF4
Solution is added in six orifice plates, and liquor capacity finally complements to 2mL, is eventually adding 2 μ L and helps and turns helping for 5~20 μ g/mL of agent and turn agent, will
It after whole system mixes, is transferred in a hole of pre-coated good 6 orifice plates, is put into after shaking up in 37 DEG C of incubators, overnight.
2) cell that yesterday infects (the 1st day) after virus infection 24 hours, is taped against preprepared feeder cells
On.
3) it the 2nd day, changes DMEM high glucose medium into human embryonic cells culture solution, continues to cultivate.Change liquid one within every 2 days later
It is secondary.
4) it observes day by day, when cell long to the 5th~7 day, it is seen that small cell aggregation, cellular morphology obviously occur
Change, growth flocks together.
5) up to the 11st day, since MEF cell uses overlong time, therefore culture solution changes CM (conditioned into
Medium, conditioned medium) the normal culture solution of+hESC (human embryo stem cell) 1:1.2 (volume ratio) mixed liquors, to be supplied to
The nutrition of cell abundance.
6) long to the 19th day to cell, clone can be chosen, the clone of embryonic stem cell sample is chosen to new MEF, is pressed
HESC (hESC) cultural method continues culture 10 days, amplification.IPS cell clone and normal human embryonic stem are thin
Born of the same parents are morphologically no different substantially.
The cell of above-mentioned acquisition is carried out to the identification (alkaline phosphatase activities detection) of iPS cell,
Multipotential stem cell has successfully been obtained as the result is shown.
The preparation of 3 multipotential stem cell of embodiment
(1) separation and sorting of total T lymphocyte
1) peripheral blood in anticoagulant tube is transferred in centrifuge tube, 400-500g (centrifugal force) is centrifuged 5-10min, centrifugation knot
Lower layer's haemocyte is transferred in 50mL centrifuge tube after beam, the physiological saline that two volumes are added is diluted；
2) a new sepmate centrifuge tube (purchased from STEM CELL company) is taken, Ficoll lymphocyte separation medium is added
Haemocyte after dilution is transferred to sepmate centrifuge tube by (being purchased from STEM CELL company) 12mL, and 600-800g is centrifuged 15-
20min；
3) after being centrifuged, supernatant liquid is poured out, physiological saline is added and carries out washing 1 time；
4) CD3 is used+Sorting kit (being purchased from STEM CELL company) sorts T lymphocyte from above-mentioned mononuclearcell
Out.
(2) preparation of feeder cells
1) the MEF cell in P2 generation (mouse embryo fibroblast stem cell) is recovered；
2) when MEF cell confluency reaches 80~90%, MEF cell is passed on.Then by 1-3 × 105It is thin
Born of the same parents are inoculated into six orifice plates；
3) processing 2 hours was carried out with the mitomycin C of 5-20 μ g/mL in 1st；
4) it changes MEF culture medium after 2 hours into, obtains feeder cells.
(3) culture of sendai virus transfection and iPS cell
1) according to sendai virus reprogramming kit operation：By the sendai virus containing OCT4, SOX2, C-MYC and KLF4
Solution is added in six orifice plates, and liquor capacity finally complements to 2mL, is eventually adding 2 μ L and helps and turns helping for 5~20 μ g/mL of agent and turn agent, will
It after whole system mixes, is transferred in a hole of pre-coated good 6 orifice plates, is put into after shaking up in 37 DEG C of incubators, overnight.
2) cell that yesterday infects (the 1st day) after virus infection 24 hours, is taped against preprepared feeder cells
On.
3) it the 2nd day, changes DMEM high glucose medium into human embryonic cells culture solution, continues to cultivate.Change liquid one within every 2 days later
It is secondary.
4) it observes day by day, when cell long to the 5th~7 day, it is seen that small cell aggregation, cellular morphology obviously occur
Change, growth flocks together.
5) up to the 12nd day, since MEF cell uses overlong time, therefore culture solution changes CM (conditioned into
Medium, conditioned medium) the normal culture solution of+hESC (human embryo stem cell) 1:1.5 (volume ratio) mixed liquors, to be supplied to
The nutrition of cell abundance.
6) long to the 20th day to cell, clone can be chosen, the clone of embryonic stem cell sample is chosen to new MEF, is pressed
HESC (hESC) cultural method continues culture 15 days, amplification.IPS cell clone and normal human embryonic stem are thin
Born of the same parents are morphologically no different substantially.
The identification (alkaline phosphatase activities detection) that the cell of above-mentioned acquisition is carried out to iPS cell, is successfully obtained as the result is shown
Obtained multipotential stem cell.
The existing preparation method induced multi-potent stem cell of comparative example 1
(1) select application on human skin fibroblast as donorcells
Application on human skin fibroblast is provided by Ji'nan University's biological medicine study base, culture medium DMEM+10%FBS；
(2) preparation of feeder cells
1) by the MEF cell recovery in P2 generation；
2) when MEF cell confluency reaches 80~90%, MEF cell is passed on.Then by 1-3 × 105It is thin
Born of the same parents are inoculated into six orifice plates；
3) processing 2 hours was carried out with the mitomycin C of 5-20 μ g/mL in 1st；
4) it changes MEF culture medium after 2 hours into, obtains feeder cells.
(3) virus transfection and reprogramming：
(contain transcription factor in four using retroviral vector：OCT-4, Sox2, c-Myc and Klf4) it transfects into fiber
Cell；
(4) cell culture after transfecting：
1) it after gene transfer 2 days, with trypsin digestion and cell and is inoculated into six orifice plates, mouse embryo is contained in six orifice plates
Tire feeder cells (MEF, murine embryonic fibroblast cells) and culture medium (DMEM+10%FBS), often
The inoculating cell density in hole is 5 × 104/mL；
2) after being inoculated with 24 hours, culture medium is changed into ESC culture medium, medium component is：Knockout DMEM+10%
FBS+10%serum replacement without LIF (LIF ELISA, leukemia inhibitory
factor)；
3) after transduceing 7 days, the celliform iPS cell clone of a small amount of ESC can be obtained.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (8)
1. a kind of method for inducing T lymphocyte at multipotential stem cell, which is characterized in that include the following steps：
T lymphocyte will be transfected containing the sendai virus of stem cell transcription factor gene, obtains infection cell；
The infection cell is mixed with feeder cells, carries out the first culture, carries out the using human embryonic stem cell medium
Two cultures, then carry out third training using the conditioned medium of feeder cells and the mixed liquor of human embryonic stem cell medium
It supports, the 4th culture is finally carried out using human embryonic stem cell medium, obtains multipotential stem cell；
First culture be specially：Infection cell is mixed with feeder cells, is cultivated using feeder cells culture medium, so
Feeder cells culture medium is abandoned afterwards；
The formula of conditioned medium is：Culture supernatant of the P2 for MEF cell.
2. the method according to claim 1, wherein the stem cell transcription factor is OCT4, SOX2, C-MYC
Or the mixture of one or more of KLF4.
3. the method according to claim 1, wherein the transfection is 24~36h of transfection under the conditions of 37 DEG C.
4. the method according to claim 1, wherein the formula of the human embryonic stem cell medium is：DMEM/
F12, serum substitute, β-thioglycol, 1% nonessential amino acid, Basic Fibroblast Growth Factor.
5. the method according to claim 1, wherein the conditioned medium and the embryonic stem cell medium
Volume ratio be 1：(1~1.5).
6. the method according to claim 1, wherein it is described transfect use help and turn agent as polybrene.
7. described second cultivates the method according to claim 1, wherein the time of first culture is 1 day
Time be 9~11 days.
8. the method according to claim 1, wherein the time of the third culture be 8~10 days, the described 4th
The time of culture is 7~15 days.
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