CN105624114B - A kind of stem cell differential medium and its application - Google Patents
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Abstract
The invention discloses a kind of stem cell differential medium and its application.Culture medium culture medium based on α MEM, adds following composition in proportion：The FBS of final concentration of percent by volume 10~20%, the BSA of final concentration of mass volume ratio 0.1~1%, final concentration of 10‑4~10‑5Mol/L mercaptoethanol, final concentration of 1~5mM l-GLUTAMINE, final concentration of 10 100ng/ml stem cell factor, final concentration of 10 50ng/ml interleukin-13, final concentration of 1~5U hematopoietin, final concentration of 10~100 μ g/ml vitamin C, final concentration of 1~20 μ g/ml lipoic acid.The culture medium can on individual cell level the 100% accurate differentiation potential for assessing hematopoietic stem/progenitor cells, and flux is high, time-consuming short.
Description
Technical field
The present invention relates to biological field, more particularly to a kind of stem cell differential medium and its application.
Background technology
The culture medium of present analysis hematopoietic stem/progenitor differentiation potential is semisolid culturemedium (MethoCultTM), principle
It is hematopoietic stem/progenitor Proliferation, Differentiation formation colony under the semi-solid medium for adding the factor containing Proliferation, Differentiation, based on this
The appraisal procedure of semisolid culturemedium is exactly classical colony forming unit experiment (ColonyForming Unit Assay).Base
A group cell mixing can only be analyzed in the Colony forming experiment analytical method of semisolid culturemedium, advantage is limited only to analysis one
Can form the type and quantity of various hematopoietic colonies in individual cell mass, but can not on individual cell level Accurate Analysis it is single
The differentiation potential of hematopoietic stem/progenitor.Although semisolid culturemedium can limit to migration of the individual cells when forming colony,
It is that individual cells differentiation is produced or multiple cells are because locus is superimposed that a mixed type colony can not be excluded in real operation
Cell mixing pollution break up together caused by.In addition, Colony forming experiment is 3.3ml using the least unit of culture medium, can
To analyze two cell mixture repeat samples, and each unit uses waste about 1.1ml semisolid culturemediums, waste rate
Up to 33%.Colony forming experiment based on semisolid culturemedium, also in the presence of following deficiency：The sticky factor of semisolid culturemedium is led
When causing operating cost, it is unfavorable for extensive batched operation；When semisolid culturemedium is drawn with pipettor or syringe
It is difficult to accurately control culture volume, so as to introduce larger error between repeat samples.
The content of the invention
It is an object of the invention to overcome the shortcoming and deficiency of existing hematopoietic stem/progenitor cells differentiation potential evaluating in vitro technology,
A kind of stem cell differential medium is provided.
Another object of the present invention is to provide the application of the stem cell differential medium.In hematopoietic stem/progenitor point
Change potential evaluating in vitro technology and apply above-mentioned stem cell differential medium, can realize 10~14 days and quickly, in high volume assess single
The differentiation potential of hematopoietic stem/progenitor cells, accurate rate 100% is not in error in judgement.
The purpose of the present invention is achieved through the following technical solutions：A kind of stem cell differential medium, composition composition is as follows：With
Culture medium based on α-MEM, adds following composition in proportion：FBS (the tire ox bloods of final concentration of percent by volume 10~20%
Clearly), the BSA (bovine serum albumin) of final concentration of mass volume ratio 0.1~1%, final concentration of 10-4~10-5Mol/L 2- β-
Mercaptoethanol (mercaptoethanol), final concentration of 1~5mM L-gln (left-handed-glutamine), final concentration of 10-
100ng/ml SCF (stem cell factor), final concentration of 10-50ng/ml IL3 (interleukin-13), final concentration of 1~5U EPO
(hematopoietin), final concentration of 10~100 μ g/ml vitamin C, final concentration of 1~20 μ g/ml (±)-α-
Lipoic acid (lipoic acid).
Described stem cell is hematopoietic stem/progenitor.
Application of the described stem cell differential medium in assessing hematopoietic stem/progenitor differentiation potential, is particularly suitable for
It is estimated applied to single hematopoietic stem/progenitor differentiation potential.
Application of the described stem cell differential medium in assessing hematopoietic stem/progenitor differentiation potential, including it is as follows
Step：
(1) described stem cell differential medium is added in Tissue Culture Plate；
(2) by flow sorter, cell to be analyzed is sorted using single cell mode, toward each hole in Tissue Culture Plate
It is sorted into a cell to be analyzed；
(3) Tissue Culture Plate containing individual cells is transferred to cell culture incubator, 37 DEG C, 5%CO2Under the conditions of culture 10~
14 days；In the training period, fresh stem cell differential medium is changed every other day, and update method is the old culture medium of removal half,
The isometric 37 DEG C of fresh preheating culture mediums of addition；
(4) cell that step (3) culture propagation is obtained is detected, determines the differentiation potential of cell to be analyzed.
Step (1)~(3) are aseptically to be operated.
Described Tissue Culture Plate is preferably 96 porocyte culture plates.
It is immune that the method for detection described in step (4) includes cell rejection tablet, cell dyeing morphological analysis and streaming
Phenotypic analysis.
Described cell dyeing morphological analysis is analyzed for Giemsa staining.
Used antibody includes the antibody of identification medullary system macrophage, identification marrow in described streaming Immunophenotype analysis
It is the antibody of granulocyte, recognizes the antibody of medullary system erythroid cells.
The present invention has the following advantages and effect relative to prior art：
1st, the culture medium that the present invention is provided can dive in the 100% accurate differentiation for assessing hematopoietic stem/progenitor cells on individual cell level
Can, it is to avoid a mixing colony is because polluting the vacation of multiple hematopoietic stem/progenitor cells in the experiment of semisolid culturemedium Colony forming
Positive phenomenon.
2nd, it is liquid culture medium that the present invention, which provides stem cell differential medium, overcomes traditional semisolid culturemedium needs
Liquefaction in advance, because it is sticky cause volume weigh it is inaccurate, draw between difficult and repeat samples because volume is inaccurate and error compared with
Big the shortcomings of.
3rd, the stem cell differential medium that the present invention is provided can carry out changing liquid at any induction differentiation moment, be easy in time
Fresh culture is updated, the optimum condition of cell proliferation and differentiation is maintained.
4th, the stem cell differential medium dosage that the present invention is provided is easily controlled, the dosage of Single cell culture
Can be in 20-50ul/ holes, with reference to 96 orifice plates, 192 orifice plates, 384 orifice plates can carry out high pass by automatic pipettor, volley of rifle fire etc.
Amount packing sample operation, greatly improves operating efficiency.
5th, culture medium of the invention contains the additive of enhancing cell viability, and single hematopoietic stem/progenitor cells increased through 10-14 days
Grow differentiation, 10000~100000 order of magnitude cells can be obtained, be easy to collect carry out rejection tablet, cell dyeing morphological analysis with
And streaming Immunophenotype analysis.
Brief description of the drawings
Fig. 1 is single hematopoiesis ancestral/stem cell Proliferation, Differentiation 14 days in the stem cell differential medium that embodiment 1 is provided
Microscope (10 ×) photo figure.
Fig. 2 is the Giemsa staining point after single hematopoiesis ancestral/stem cell breeds 14 days in the culture medium prescription of embodiment 1
Photo (40 ×) figure of analysis；Wherein, arrow represents medullary system granulocyte, and square frame represents medullary system monocyte.
Fig. 3 is the streaming immunophenotype after single hematopoiesis ancestral/stem cell breeds 14 days in the culture medium that embodiment 1 is provided
Analysis result figure；Wherein, figure (A) shows that the cell of Proliferation, Differentiation contains medullary system (Mac1+, Gr1+) cell, and figure (B) shows propagation
The cell of differentiation contains red system's (TER119+) cell.
Fig. 4 is the Giemsa staining point after single hematopoiesis ancestral/stem cell breeds 14 days in the culture medium prescription of embodiment 2
Photo (40 ×) figure of analysis；Wherein, Ery represents red blood cell, and Mac represents macrophage, and Gran represents granulocyte.
Fig. 5 is the flow cytometer showed qualification figure of Immunophenotyping；Wherein, 1 is that embodiment 3 breeds the cell obtained；2 be outer
All blood monocytes, are used as check analysis；Scheme the CD11b antibody staining flow cytometer showeds of (A) for identification myeloid cell surface molecular
Figure, figure (B) is identification medullary system surfaces of granulocytes molecule Gr1 antibody staining flow cytometer showed figure.
Fig. 6 is hematopoiesis ancestral/stem cell respectively under the stem cell differential medium culture that embodiment 3 and comparative example 1 are provided
Proliferation, Differentiation result figure；Scheme the stem cell differential medium that (A) provides for comparative example 1, figure (B) is doing that embodiment 3 is provided
Cell differential medium.
Fig. 7 is hematopoiesis ancestral/stem cell respectively under the stem cell differential medium culture that embodiment 3 and comparative example 1 are provided
Propagation yield statistical results chart.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment 1
A kind of precise formulations of stem cell differential medium are as follows：Under whole process aseptic condition, with commercialized α-MEM
Based on culture medium, following composition is added in proportion：10%FBS (hyclone), 0.1%BSA (bovine serum albumin), 1 × 10- 5M 2- β-mercaptoethanol (mercaptoethanol), 1mM L-gln (left-handed-glutamine), 10ng/ml SCF (stem cells
The factor), 10ng/ml IL3 (interleukin-13), 1U EPO (hematopoietin), 10 μ g/ml vitamin Cs, 1 μ g/ml (±)-
α-Lipoic acid (lipoic acid).
Application method:
(1) automatic sample-adding system or the volley of rifle fire are utilized under aseptic condition, with the volume in 50 μ l/ holes by the culture of the present embodiment
Base is transferred to 96 orifice plates.
(2) cell to be analyzed is sorted using single cell mode, each hole is sorted into by flow sorter under aseptic condition
One hematopoietic stem/progenitor.
(3) 96 orifice plates containing individual cells are transferred to cell culture incubator, 37 DEG C, 5%CO under aseptic condition2Under the conditions of train
Support 14 days.
(4) during step (3) culture, fresh stem cell differential medium is updated every other day, and update method is removal half
Old culture medium, the isometric fresh 37 DEG C of preheatings culture medium of addition.
(5) the micro- each hole cell proliferative conditions of Microscopic observation under aseptic condition, the cell of each hole propagation is all from list
One initiator cell.Microscopy results are as shown in figure 1, show that single HPC is induced in the culture medium of the present embodiment
Differentiation 14 days, cell is largely bred.
(6) cell in each hole is collected, takes half to carry out cell rejection tablet and (the method reference of Switzerland-Giemsa staining
Hematopoietic differentiation.Kyung-Dal Choi,Maxim Vodyanik,and Igor
ISlukvin.June 10,2012.StemBook [Internet]), cytomorphology identification, amplification 400 are carried out under microscope
Take pictures again.As a result as shown in Fig. 2 being clearly visible monocyte, granulocyte, monocyte is shown, granulocyte is all by one
Single common progenitor cell is differentiated, so as to prove that the single progenitor cells initially analyzed are Meloid progenitors.
(7) collect the cell in each hole, take half carry out antibody staining (method refers to Kiel MJ, Yilmaz OH,
Iwashita T,Terhorst C,Morrison SJ.SLAM Family Receptors
DistinguishHematopoietic Stem and Progenitor Cells and Reveal Endothelial
Niches for StemCells.Cell.2005；121:1109-1121.), selected antibody includes identification medullary system monocyte
Antibody (CD11b, article No. 11-0112-82, ebioscience), antibody (Gr1, article No. 12-5931- for recognizing medullary system granulocyte
82, ebioscience), the pedigree such as antibody (TER119, article No. 17-5921-82, Ebioscience) of identification erythroid cells is thin
Born of the same parents' type, carries out flow cytometer showed, the progeny cell type that the unicellular propagation of accurate identification is produced.Result as shown in Figure 3 shows
Single HPC Multiplying culture in the present embodiment culture medium carries out cytomorphology detection and finds Proliferation, Differentiation after 14 days
For monocyte and granulocyte, so as to prove that single HPC to be analyzed is multipotency Meloid progenitor.
Embodiment 2
Under whole process aseptic condition, the culture medium based on commercialized α-MEM adds following composition in proportion:
20%FBS (hyclone), 1%BSA (bovine serum albumin), 1 × 10-4M 2- β-mercaptoethanol (mercaptoethanol),
5mM L-gln (left-handed-glutamine), 100ng/ml SCF (stem cell factor), 50ng/ml IL3 (interleukin-13), 5U EPO
(hematopoietin), 100 μ g/ml vitamin Cs, 20 μ g/ml (±)-α-Lipoic acid (lipoic acid).
Application method is described with example 1, analyzes the differentiation potential of single HPC.As a result as shown in figure 4, to
The cell of 14 days carries out Morphological Identification after the differentiation culture of the present embodiment culture medium, and discovery has erythroblast (Ery), macrophage thin
Born of the same parents (MAC), granulocyte (Gran), so as to reversely prove that individual cells initially to be analyzed are Hematopoietic Stem/ancestrals of many differentiation potentials
Cell.
Embodiment 3
Under whole process aseptic condition, the culture medium based on commercialized α-MEM adds following composition in proportion：
10%FBS (hyclone), 0.5%BSA (bovine serum albumin), 5 × 10-4M 2- β-mercaptoethanol (mercaptoethanol),
2mM L-gln (left-handed-glutamine), 50ng/ml SCF (stem cell factor), 25ng/ml IL3 (interleukin-13), 3U EPO
(hematopoietin), 50 μ g/ml vitamin Cs, 10 μ g/ml (±)-α-Lipoic acid (lipoic acid).
Application method is with example 1, and the differentiation for analyzing a unknown HPC using the culture medium of the present embodiment is dived
Power, flow cytometer showed is identified (such as Fig. 5 institutes
Show) show that in the cell mass bred be myeloid cell (CD11b+, Gr1+) entirely, so as to deduce the unknown hematopoiesis ancestral
Cell is grain-monokaryon common progenitor cell GMP.
Comparative example 1
The culture medium of the stem cell of this comparative example 1 differs only in not lipoic acid with the culture medium of embodiment 3.
Application method be the same as Example 1.By counting 100 single hematopoietic stem/progenitor cells are trained under conditions of lipoic acid is whether there is
Support 14 days, cell quantity is that Proliferation, Differentiation fails less than 50.Fig. 6 illustrates there is 60% in the case of no lipoic acid (Fig. 6 A)
Single hematopoietic stem/progenitor cells are unable to Proliferation, Differentiation success, and lipoic acid then only has 10% single to make in the case of there is (Fig. 6 B)
Blood stem/progenitor cells are unable to Proliferation, Differentiation success.In Fig. 7, without lipoic acid in the case of individual cells differentiation and proliferation 14 days it is average only
The order of magnitude of hundred cells can be reached, ten thousand cell number magnitudes (n=10, P can be then reached in the case of having lipoic acid<
0.01) the propagation yield of cell can be dramatically increased by, illustrating the presence of lipoic acid in culture media composition of the present invention.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
1. a kind of stem cell differential medium, it is characterised in that be made up of following composition：The culture medium based on α-MEM, by than
The following composition of example addition：The FBS of final concentration of percent by volume 10%, the BSA of final concentration of mass volume ratio 0.5%, end are dense
Spend for 5 × 10-4Mol/L mercaptoethanol, 2mM left-handed-glutamine, final concentration of 50ng/ml stem cell factor, end are dense
Spend interleukin-13 for 25ng/ml, it is final concentration of 3U hematopoietin, final concentration of 50 μ g/ml vitamin C, dense eventually
Spend for 10 μ g/ml lipoic acid；
Described stem cell is hematopoietic stem/progenitor.
2. application of the stem cell differential medium in assessing hematopoietic stem/progenitor differentiation potential described in claim 1.
3. stem cell differential medium according to claim 2 answering in assessing hematopoietic stem/progenitor differentiation potential
With, it is characterised in that：The number of described hematopoietic stem/progenitor is 1.
4. the answering in assessing hematopoietic stem/progenitor differentiation potential of the stem cell differential medium described in Claims 2 or 3
With, it is characterised in that comprise the following steps：
(1) described stem cell differential medium is added in Tissue Culture Plate；
(2) by flow sorter, cell to be analyzed is sorted using single cell mode, toward each hole sorting in Tissue Culture Plate
Enter a cell to be analyzed；
(3) Tissue Culture Plate containing individual cells is transferred to cell culture incubator, 37 DEG C, 5%CO2Under the conditions of cultivate 10~14 days；
In the training period, fresh stem cell differential medium is changed every other day, and update method is the old culture medium of removal half, addition etc.
37 DEG C of fresh preheating culture mediums of volume；
(4) cell that step (3) culture propagation is obtained is detected, determines the differentiation potential of cell to be analyzed.
5. stem cell differential medium according to claim 4 answering in assessing hematopoietic stem/progenitor differentiation potential
With, it is characterised in that：Described Tissue Culture Plate is 96 porocyte culture plates.
6. stem cell differential medium according to claim 4 answering in assessing hematopoietic stem/progenitor differentiation potential
With, it is characterised in that：The method of detection described in step (4) includes cell rejection tablet, cell dyeing morphological analysis and stream
Formula Immunophenotype analysis.
7. stem cell differential medium according to claim 4 answering in assessing hematopoietic stem/progenitor differentiation potential
With, it is characterised in that：Described cell dyeing morphological analysis is analyzed for Giemsa staining.
8. stem cell differential medium according to claim 4 answering in assessing hematopoietic stem/progenitor differentiation potential
With, it is characterised in that：In described streaming Immunophenotype analysis used antibody include identification medullary system macrophage antibody,
Recognize the antibody of medullary system granulocyte, recognize the antibody of medullary system erythroid cells.
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| CN103070161A (en) * | 2013-01-09 | 2013-05-01 | 广州爱菲科生物科技有限公司 | Cryopreservation liquid and cryopreservation method for adipose tissue-derived mesenchymal stem cells ( ADSC) |
| CN104911149A (en) * | 2015-06-15 | 2015-09-16 | 中国医学科学院输血研究所 | Method for establishing differentiation model from human embryo stem cells to eosinophils |
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| CN103070161A (en) * | 2013-01-09 | 2013-05-01 | 广州爱菲科生物科技有限公司 | Cryopreservation liquid and cryopreservation method for adipose tissue-derived mesenchymal stem cells ( ADSC) |
| CN104911149A (en) * | 2015-06-15 | 2015-09-16 | 中国医学科学院输血研究所 | Method for establishing differentiation model from human embryo stem cells to eosinophils |
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| Title |
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| FACSAria流式细胞仪96 孔微孔板单个细胞分选方法的优化和应用;刘锡娟等;《生物学通报》;20100320;第45卷(第3期);第51-55页,主要涉及摘要 * |
| Properties of hematopoietic stem/progenitor cells derived from human placenta tissues;Zhang Tao等;《Journal of Clinical Rehabilitative Tissue Engineering Research》;20080520;第12卷(第21期);第4172-4176页，主要涉及摘要、第4173页右栏第2段 * |
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