CN101314767A - Culture solution for reinforcing expression of fetal liver hemopoietic stem cell CD34 and preparation thereof - Google Patents

Culture solution for reinforcing expression of fetal liver hemopoietic stem cell CD34 and preparation thereof Download PDF

Info

Publication number
CN101314767A
CN101314767A CNA200810040136XA CN200810040136A CN101314767A CN 101314767 A CN101314767 A CN 101314767A CN A200810040136X A CNA200810040136X A CN A200810040136XA CN 200810040136 A CN200810040136 A CN 200810040136A CN 101314767 A CN101314767 A CN 101314767A
Authority
CN
China
Prior art keywords
stem cell
human
factor
fetal liver
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CNA200810040136XA
Other languages
Chinese (zh)
Inventor
郝晓南
赵珺
章永泰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SHANGHAI TIANSHENG BIOTECHOLOGY CO Ltd
Original Assignee
SHANGHAI TIANSHENG BIOTECHOLOGY CO Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SHANGHAI TIANSHENG BIOTECHOLOGY CO Ltd filed Critical SHANGHAI TIANSHENG BIOTECHOLOGY CO Ltd
Priority to CNA200810040136XA priority Critical patent/CN101314767A/en
Publication of CN101314767A publication Critical patent/CN101314767A/en
Priority to PCT/CN2009/000244 priority patent/WO2010000125A1/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/14—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention relates to a culture solution of strengthening fetal liver hematopoietic stem cell CD34 expression and a preparation method thereof and belongs to the biological engineering technology field. The components and the weight percentage of the culture solution are as follows: 56.5-64.7 percent of MEM; 29.4-30.4 percent of RPMI-1640; and 5.88-13.0 percent of Hanks etc. The preparation method comprises the following steps: (1)preparing the basic culture solution;(2) selecting again relative components according to the weight percentage, adding the same to the basic culture solution in sequence; agitating and dissolving; (3) purifying a mesenchymal cell, putting in a culture bottle, culturing, washing, mixing, continuously culturing, collecting supernatant fluid, filtering, centrifuging, removing all cell fragments and non-soluble matters, and obtaining a conditioned culture solution; (4) mixing the conditioned culture solution with MEM, utilizing sodium bicarbonate to adjust the mixed culture solution to pH of 7.40 to 7.45, filtering and obtaining the culture solution. The culture solution strengthens expression of the CD34 expression characteristics of the stem cell. The stem cell reaches an identifiable and purified range, the identified quantity of the stem cell is improved, the culture solution is safe and reliable, and the clinical application is satisfied.

Description

Strengthen nutrient solution of expression of fetal liver hemopoietic stem cell CD 34 and preparation method thereof
Technical field
The present invention relates to nutrient solution of a kind of technical field of bioengineering and preparation method thereof.Be specifically related to a kind of nutrient solution that strengthens expression of fetal liver hemopoietic stem cell CD 34 and preparation method thereof.
Background technology
Hemopoietic stem cell is suggested and is applied to the clinical treatment potentiality that demonstrated it at the beginning of last century, many diseases in the blood system make the clinical symptom of sufferer be improved behind cellular replacement therapy, and survival time of patients prolongs significantly.The main source of hemopoietic stem cell comprises bone marrow hemopoietic stem cell, umbilical hemopoietic stem cell, fetal liver hemopoietic stem cell.Because the hemopoietic stem cell source is different, the antigenicity that stem cell possessed is also different, mainly show as: the antigenicity of bone marrow hemopoietic stem cell (for example: HLA) strong, the bone marrow hemopoietic stem cell that implants, if without joining type, at first host's immunity system can produce repulsive interaction to the hemopoietic stem cell that implants.The strong stem cell of antigenicity of input also can repel the host, and the intensive rejection can cause a series of systematic allergic immune reaction, and causes the dysfunction and the depletion of human organ.Therefore, after the implantation of bone marrow hemopoietic stem cell must be done the antigenic type of donor and part fully relatively, just can do the treatment that cell is implanted finding out donor and host antigen " identical " or the utmost point " close " person, this finally cause the patient have only 100,000 or millionth chance search out the donor that meets self cell requirement, there is greatly difficulty in clinical application.
The hemopoietic stem cell that Cord blood provides since antigenicity than the bone marrow hemopoietic stem cell a little less than, so umbilical hemopoietic stem cell is the stem cell that ideal has clinical treatment value in theory, its body rejection is light than the bone marrow hemopoietic stem cell.Because the limited amount of umbilical hemopoietic stem cell, can not satisfy adult and big-age-child patient and implant the clinical requirement that stem cell is repaired body, because the umbilical cord blood hematopoietic stem cell in-vitro multiplication, remain at many key technical problems unresolvedly, clinical practice is used and is had difficulties.Compare with the above two, fetal liver hemopoietic stem cell has following advantage.The first, the hemopoietic stem cell antigenicity than umbilical hemopoietic stem cell a little less than, so that stem cell antigen is joined type is simple and easy relatively and obtain in small-scale people tissue donor easily; The second, the fetal liver hemopoietic tissue contains and has a considerable amount of stem cells (50～120 * 10 6), and umbilical hemopoietic stem cell only has 1～2 * 10 6, the patient that the umbilical hemopoietic stem cell of this quantity only can satisfy body weight≤20 kilogram implants requirement; The 3rd, the surviving rate height of stem cell divides voltinism strong.The cell colony that unit cell produces all is higher than marrow and navel haematogenous hemopoietic stem cell, and the stem cell that is purified into only needs ABO, HLA, and type is joined in six sites, just can be actually used in the patient, and it joins the type probalility of success can be up to 25%.
Find by retrieval, Chinese patent application number: 02134314.4, publication number: CN1389566, title: nerve stem cell culture medium and preparation method thereof, this technology readme is: disclose a kind of nerve stem cell culture medium and preparation method thereof, by DMEM and F12 by the basic culture solution that mixes at 1: 1, Regular Insulin, the hydrochloric acid butanediamine, sodium selenide, hydrocortisone, L-glutaminate, human transferrin and Progesterone are formulated, have directed growth of different sources seed cell such as the myeloid tissue of making and be divided into the neural stem cell function, can be at any time provide the relevant neural stem cell that needs for medical research teaching and clinical application like clockwork.Be subjected to the function influence of microenvironment bigger because the CD34 of hemopoietic stem cell expresses, a little less than some stem cell CD34 in partial periodicity expresses extremely, and some stem cell CD34 in the same cycle expresses very strong (this phenomenon is called " drift is expressed " again).This biological nature uses present purification process to express strong stem cell (the CD34 cell being carried out methods such as mark by fluorescent-labeled antibody or CD34 separating kit) by purifying CD34, and CD34 expresses weak stem cell in one-period, owing to can not then be left in the basket by significant notation, this biological nature of stem cell (CD34 expression) makes present purification process to carry out purifying to those stem cells through sign, but make stem cell purifying quantity instability, and the hemopoietic stem cell that can not effectively be purified into capacity is applied to clinical, for the patient provides the repeatedly chance of treatment.
This technical role is to have a directed cell culture medium that is divided into the neural stem cell function of growing of different sources seed cell such as myeloid tissue made from a kind of.There is uncertainty in cell in the atomization because neural stem cell is grown in orientation, and whether the cell after differentiation is inherited and had former cells and characteristic of stem and still can not determine, so there is bigger risk in clinical application.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of nutrient solution that strengthens expression of fetal liver hemopoietic stem cell CD 34 and preparation method thereof is provided.Nutrient solution of the present invention, its effect are that CD34 expression characteristic that stem cell is had is implemented to strengthen and expressed, reach can identify, the scope of purifying, can identify quantity thereby greatly improved stem cell; Preparation method of the present invention makes the stem cell a little less than some CD34 expresses in the script hemopoietic stem cell, displays again, make more the displaying of original hemopoietic stem cell, significantly improved hemopoietic stem cell purifying quantity, safe and reliable, satisfy clinical application.
The nutrient solution of above-mentioned enhancing expression of fetal liver hemopoietic stem cell CD 34 involved in the present invention, its component and weight percent are:
MEM：56.5％-64.7％；
RPMI-1640：29.4％-30.4％；
Hanks：5.88％-13.0％；
Human stem cell factor-2:470 * 10 -9%-1740 * 10 -9%;
Human stem cell factor: 1.9 * 10 -9%-2.8 * 10 -9%;
Human granulocyte-macrophage colony stimutaing factor: 2.0 * 10 -9%-2.9 * 10 -9%;
CD135 ligandin: 9.4 * 10 -9%-10.8 * 10 -9%;
Human erythropoietin: 1.4 * 10 -9%-2.4 * 10 -9%;
Human epithelial cell generates the factor: 9.4 * 10 -9%-10.8 * 10 -9%;
Human short fibroblast growth factor-b:9.4 * 10 -9%-10.8 * 10 -9%.
The term of said components of the present invention and symbol specify as follows:
Described MEM is meant: minimum essential medium substratum, by the Zooblast culture medium of U.S. GIBCO company production, contain L-glutaminate, do not contain bicarbonate of ammonia, Sodium.alpha.-ketopropionate, 2 ℃ of-8 ℃ of preservations are more suitable for supporting the growth of various mammalian cells.Existing open source literature record: 1, Zhang Yingchun, Liao Xiaomei etc., " different substratum are to the influence of hippocampal slices activity and microtubule-associated protein tau expression ", Chinese Academy of Medical Sciences's journal, middle figure classification number: R745.7 Document code: A article numbering: 1000-503X (2005) 04-0513-05; 2, Han Ming, Dong Liping, Yuan Fang, Earle ' s liquid and MEM substratum are to the influence [J] of neuronal activity, Chinese rehabilitation theory and practice, 2005,11 (2): 125-126.
Described RPMI-1640, be meant: the RPMI-1640medium substratum, be used to cultivate the liquid nutrient medium of normal human leukocyte by Roswell ParkMemorial Institute exploitation, contain L-glutaminate (L-Glutamine), do not contain bicarbonate of ammonia (HEPES), 2 ℃ of-8 ℃ of preservations.Existing open source literature record: 1, Huang Bei, Li Zhengang etc., " the RPMI1640 substratum is to the preliminary observation of hydra body surface osmotrophy ", the zoology magazine, middle figure classification number: Q172, Document code: A, the article numbering: 025023263 (2001) 06240203, calendar year 2001; 2, E Zheng etc., " tissue culture technique ", Beijing: People's Health Publisher, 1985.219.
Described Hanks, be meant: balanced salt buffered soln (GENMED Hanks), be a kind of cleaning or the most frequently used solution of groupization pre-treatment that is intended to be used for cell and tissue, the achievement that its stdn component prescription is delivered in 1949 according to Hanks and Wallace, and meet the regulation that U.S. vitro tissue is cultivated the stdn council (Tissue Culture Standards Committee, In Vitro).The place of production U.S..Product promptly arrives i.e. usefulness, and is strict aseptic, stable performance.The Hanks balanced salt solution (HBSS) of standard contains Repone K, sodium-chlor, potassium primary phosphate, Sodium phosphate dibasic, D-glucose etc., but not calcic, magnesium ion.Existing open source literature record: " the non-procedural DNA synthetic test of State Standard of the People's Republic of China GB 15193-10-94 ".
Human stem cell factor-2, full name is: human Stem Cell Growth Factor-2; Human stem cell factor, full name is: human Stem Cell Growth Factor; Human granulocyte-macrophage colony stimutaing factor, full name is: human Granulocyte Macrophage-ColonyStimulation Factor; The CD135 ligandin, full name is: human flt3 ligand; Human erythropoietin, full name is: human Erythropoietin; Human epithelial cell generates the factor, and full name is: human Epithelial Growth Factor; Human short fibroblast growth factor-b, full name is: human Fibroblast Growth Factor-b; Above-mentioned term is all on the books at open source literature.
The preferred weight percent of component of the present invention is:
MEM：59.36％-61％；
RPMI-1640：29.7％-30.14％；
Hanks：9.3％-10.5％；
Human stem cell factor-2:850 * 10 -9%-1260 * 10 -9%;
Human stem cell factor: 2.1 * 10 -9%-2.5 * 10 -9%;
Human granulocyte-macrophage colony stimutaing factor: 2.2 * 10 -9%-2.6 * 10 -9%;
CD135 ligandin: 9.8 * 10 -9%-10.4 * 10 -9%;
Human erythropoietin: 1.7 * 10 -9%-2.1 * 10 -9%;
Human epithelial cell generates the factor: 9.7 * 10 -9%-10.4 * 10 -9%;
Human short fibroblast growth factor-b:9.6 * 10 -9%-10.4 * 10 -9%.
The preparation method of the nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 involved in the present invention, make the stem cell a little less than some CD34 expresses in the script hemopoietic stem cell, again display, promote the CD34 ability to express of stem cell nature, CD34 in the tire liver is expressed weak hemopoietic stem cell give expression to the level that can be picked out by fluorescent-labeled antibody.And in the original stem cell CD34 to express strong stem cell unaffected, keep its original CD34 expression level.By cell " signal " regulation system,, cause cell to strengthen the CD34 protein expression with inactive CD34 gene activation.Therefore the present invention carries out cytodifferentiation and propagation to hemopoietic stem cell, but makes more the displaying of original hemopoietic stem cell, can significantly improve the purifying quantity of hemopoietic stem cell, satisfies clinical application.
The nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 of the present invention can make the stem cell a little less than CD34 expresses strengthen expression in the specific cycle, and its CD34 protein expression is reached can be by the sign scope.With the fetal liver hemopoietic stem cell is example, and with conventional fluorescence antibody labelling method, but the quantitative range of the CD34 fetal liver hemopoietic stem cell of mark is 10～20 * 10 6, and after using " fetal liver hemopoietic stem cell CD 34 strengthens the nutrient solution of the expressing " processing and purifying of the present invention's preparation, but the mark quantity of fetal liver hemopoietic stem cell is improved more than 5 times, can mark hemopoietic stem cell quantity be 80～100 * 10 6
The preparation method of the nutrient solution of above-mentioned enhancing expression of fetal liver hemopoietic stem cell CD 34 involved in the present invention may further comprise the steps:
1. after getting MEM, RPMI 1640, Hanks mixing in described ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after the standard test of 19-20 week,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 10-14 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T-75 type culturing bottle with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium.
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with pH regulator to 7.40～7.45 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron filter filtrations and strengthen the expression nutrient solution.
Nutrient solution of the present invention, its effect is that the CD34 expression characteristic that stem cell is had implements to strengthen expression, the CD34 expression characteristic that stem cell had that originally promptly has the CD34 expression characteristic is strengthened, reach can identify, the scope of purifying, thereby greatly improved stem cell and can identify quantity, owing to do not have cell differentiation procedure, so stem cell identifies, purifying is safe and reliable, has overcome the probabilistic shortcoming of the cytodifferentiation that exists in the prior art.
The preparation method of nutrient solution of the present invention, make the stem cell a little less than some CD34 expresses in the script hemopoietic stem cell, again display, promote the CD34 ability to express of stem cell nature, CD34 in the tire liver is expressed weak hemopoietic stem cell give expression to the level that can be picked out by fluorescent-labeled antibody.And in the original stem cell CD34 to express strong stem cell unaffected, keep its original CD34 expression level.By cell " signal " regulation system, with inactive CD34 genetic expression cell-stimulating, therefore the present invention carries out cytodifferentiation and propagation to hemopoietic stem cell, but make more the displaying of original hemopoietic stem cell, can significantly improve hemopoietic stem cell purifying quantity, satisfy clinical application.
Embodiment
Below embodiments of the invention are elaborated: present embodiment has provided detailed embodiment being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.
Embodiment one:
The nutrient solution that present embodiment strengthens expression of fetal liver hemopoietic stem cell CD 34 is that the set of dispense ratio is under the situation of 1 liter (L) in gross weight:
RPMI-1640?250ml；
Hanks?110ml；
Human stem cell factor 24 μ g;
Human stem cell factor 16ng;
Human granulocyte-macrophage colony stimutaing factor 17ng;
CD135 ligandin 92ng;
Human erythropoietin 12ng;
Human epithelial cell generates factor 92ng;
Human short fibroblast growth factor-b 92ng;
The MEM surplus.
1. after getting MEM, RPMI-1640, Hanks mixing according to the above ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after 19 all standard tests,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 13 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T 75 type culturing bottles with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron (μ M) filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with the pH regulator to 7.42 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron (μ M) membrane filtrations and strengthen the expression nutrient solution.
Implementation result: use " fetal liver hemopoietic stem cell CD 34 strengthens the nutrient solution of expressing " of method for preparing, but can make the mark quantity of fetal liver hemopoietic stem cell be increased to 80 * 10 6
Embodiment two
The nutrient solution that present embodiment strengthens expression of fetal liver hemopoietic stem cell CD 34 is that the set of dispense ratio is under the situation of 1L in gross weight:
RPMI-1640?300ml；；
Hanks?70ml；
Human stem cell factor-220 μ g;
Human stem cell factor 32ng;
Human granulocyte-macrophage colony stimutaing factor 33ng;
CD135 ligandin 108ng;
Human erythropoietin 28ng;
Human epithelial cell generates factor 108ng;
Human short fibroblast growth factor-b 108ng;
The MEM surplus.
1. after getting MEM, RPMI-1640, Hanks mixing according to the above ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after 19 all standard tests,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 13 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T-75 type culturing bottle with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron (μ M) filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with the pH regulator to 7.42 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron (μ M) membrane filtrations and strengthen the expression nutrient solution.
Implementation result: use " fetal liver hemopoietic stem cell CD 34 strengthens the nutrient solution of expressing " of method for preparing, but can make the mark quantity of fetal liver hemopoietic stem cell be increased to 85 * 10 6
Embodiment three
The nutrient solution that present embodiment strengthens expression of fetal liver hemopoietic stem cell CD 34 is that the set of dispense ratio is under the situation of 1L in gross weight:
RPMI-1640?301ml；
Hanks?98ml；
Human stem cell factor-2 13 μ g;
Human stem cell factor 25ng;
Human granulocyte-macrophage colony stimutaing factor 30ng;
CD135 ligandin 101ng;
Human erythropoietin 21ng;
Human epithelial cell generates factor 101ng;
Human short fibroblast growth factor-b 101ng;
The MEM surplus.
1. after getting MEM, RPMI-1640, Hanks mixing according to the above ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after 19 all standard tests,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 13 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T-75 type culturing bottle with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron (μ M) filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with the pH regulator to 7.42 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron (μ M) membrane filtrations and strengthen the expression nutrient solution.
Implementation result: use " fetal liver hemopoietic stem cell CD 34 strengthens the nutrient solution of expressing " of method for preparing, but can make the mark quantity of fetal liver hemopoietic stem cell be increased to 90 * 10 6
Embodiment four
The nutrient solution that present embodiment strengthens expression of fetal liver hemopoietic stem cell CD 34 is that the set of dispense ratio is under the situation of 1L in gross weight:
RPMI-1640?285ml；
Hanks?130ml；
Human stem cell factor-26 μ g;
Human stem cell factor 19ng;
Human granulocyte-macrophage colony stimutaing factor 21ng;
CD135 ligandin 96ng;
Human erythropoietin 16ng;
Human epithelial cell generates factor 96ng;
Human short fibroblast growth factor-b 96ng;
The MEM surplus.
1. after getting MEM, RPMI-1640, Hanks mixing according to the above ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after 20 all standard tests,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 12 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T-75 type culturing bottle with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron (μ M) filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with the pH regulator to 7.43 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron (μ M) membrane filtrations and strengthen the expression nutrient solution.
Implementation result: use " fetal liver hemopoietic stem cell CD 34 strengthens the nutrient solution of expressing " of method for preparing, but can make the mark quantity of fetal liver hemopoietic stem cell be increased to 89 * 10 6
Embodiment five
The nutrient solution that present embodiment strengthens expression of fetal liver hemopoietic stem cell CD 34 is that the set of dispense ratio is under the situation of 1L in gross weight:
RPMI-1640?325ml；
Hanks?50ml；
Human stem cell factor-216 μ g;
Human stem cell factor 27ng;
Human granulocyte-macrophage colony stimutaing factor 29ng;
CD135 ligandin 104ng;
Human erythropoietin 24ng;
Human epithelial cell generates factor 104ng;
Human short fibroblast growth factor-b 104ng;
The MEM surplus.
1. after getting MEM, RPMI-1640, Hanks mixing according to the above ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after 20 all standard tests,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 12 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T-75 type culturing bottle with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron (μ M) filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with the pH regulator to 7.43 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron (μ M) membrane filtrations and strengthen the expression nutrient solution.
Implementation result: use " fetal liver hemopoietic stem cell CD 34 strengthens the nutrient solution of expressing " of method for preparing, but can make the mark quantity of fetal liver hemopoietic stem cell be increased to 91 * 10 6
Embodiment six
The nutrient solution that present embodiment strengthens expression of fetal liver hemopoietic stem cell CD 34 is that the set of dispense ratio is under the situation of 1L in gross weight:
RPMI-1640?299ml；
Hanks?102ml；
Human stem cell factor-2 12 μ g;
Human stem cell factor 23ng;
Human granulocyte-macrophage colony stimutaing factor 28ng;
CD135 ligandin 99ng;
Human erythropoietin 19ng;
Human epithelial cell generates factor 99ng;
Human short fibroblast growth factor-b 99ng;
The MEM surplus.
1. after getting MEM, RPMI-1640, Hanks mixing according to the above ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after 20 all standard tests,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 12 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T-75 type culturing bottle with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron (μ M) filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with the pH regulator to 7.43 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron (μ M) membrane filtrations and strengthen the expression nutrient solution.
Implementation result: use " fetal liver hemopoietic stem cell CD 34 strengthens the nutrient solution of expressing " of method for preparing, but can make the mark quantity of fetal liver hemopoietic stem cell be increased to 93 * 10 6
Embodiment seven
The nutrient solution that present embodiment strengthens expression of fetal liver hemopoietic stem cell CD 34 is that the set of dispense ratio is under the situation of 1L in gross weight:
RPMI-1640?293ml；
Hanks?114ml；
Human stem cell factor-2 9 μ g;
Human stem cell factor 21ng;
Human granulocyte-macrophage colony stimutaing factor 24ng;
CD135 ligandin 98ng;
Human erythropoietin 18ng;
Human epithelial cell generates factor 98ng;
Human short fibroblast growth factor-b 98ng;
The MEM surplus.
1. after getting MEM, RPMI-1640, Hanks mixing according to the above ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after 19 all standard tests,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 13 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T-75 type culturing bottle with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron (μ M) filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with the pH regulator to 7.45 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron (μ M) membrane filtrations and strengthen the expression nutrient solution.
Implementation result: use " fetal liver hemopoietic stem cell CD 34 strengthens the nutrient solution of expressing " of method for preparing, but can make the mark quantity of fetal liver hemopoietic stem cell be increased to 98 * 10 6
Embodiment eight
The nutrient solution that present embodiment strengthens expression of fetal liver hemopoietic stem cell CD 34 is that the set of dispense ratio is under the situation of 1L in gross weight:
RPMI-1640?313ml；
Hanks?74ml；
Human stem cell factor-2 11 μ g;
Human stem cell factor 23ng;
Human granulocyte-macrophage colony stimutaing factor 26ng;
CD135 ligandin 102ng;
Human erythropoietin 22ng;
Human epithelial cell generates factor 102ng;
Human short fibroblast growth factor-b 102ng;
The MEM surplus.
1. after getting MEM, RPMI-1640, Hanks mixing according to the above ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after 19 all standard tests,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 13 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T-75 type culturing bottle with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron (μ M) filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with the pH regulator to 7.45 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron (μ M) membrane filtrations and strengthen the expression nutrient solution.
Implementation result: use " fetal liver hemopoietic stem cell CD 34 strengthens the nutrient solution of expressing " of method for preparing, but can make the mark quantity of fetal liver hemopoietic stem cell be increased to 99 * 10 6
Embodiment nine
The nutrient solution that present embodiment strengthens expression of fetal liver hemopoietic stem cell CD 34 is that the set of dispense ratio is under the situation of 1L in gross weight:
RPMI-1640?300ml；
Hanks?100ml；
Human stem cell factor-2 11 μ g;
Human stem cell factor 24ng;
Human granulocyte-macrophage colony stimutaing factor 29ng/l
CD135 ligandin 100ng;
Human erythropoietin 20ng;
Human epithelial cell generates factor 100ng;
Human short fibroblast growth factor-b 100ng;
The MEM surplus.
1. after getting MEM, RPMI-1640, Hanks mixing according to the above ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. get the human tire liver after 19 all standard tests,,, get 10 according to the reading of counter with the mesenchymal cell that is purified into the fetal liver mesenchyma cellular segregation 7Individual fetal liver mesenchyma cell is inserted T-75 type culturing bottle, add DMEM nutrient solution (DMEM nutrient solution, Dulbecco ' s modified Eagle ' s medium) and 2% human serum cultivate, cultivation through 13 days, supernatant liquor is extracted out, the fetal liver mesenchyma cell in the T-75 type culturing bottle is washed secondary with Hanks.Add DMEM nutrient solution and 5% human serum then and mix in T-75 type culturing bottle with the fetal liver mesenchyma cell, (condition control required: 37 ℃ of temperature, CO through 48 hours cultured continuously 2Concentration 5%), supernatant liquor is all collected.After supernatant liquor filters through 0.2 micron (μ M) filter, through the high speed centrifugation of 2500g the material of whole cell debriss and non-solubility is removed again, made conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize sodium bicarbonate (0.1 μ mol NaHCO 3) with the pH regulator to 7.45 of mixed-culture medium, can make stem cell CD 34 through twice 0.2 micron (μ M) membrane filtrations and strengthen the expression nutrient solution.
Implementation result: use the nutrient solution of method for preparing, but can make the mark quantity of fetal liver hemopoietic stem cell be increased to 100 * 10 6

Claims (10)

1, a kind of nutrient solution that strengthens expression of fetal liver hemopoietic stem cell CD 34 is characterized in that, component and weight percent are:
MEM：56.5％-64.7％；
RPMI-1640：29.4％-30.4％；
Hanks：5.88％-13.0％；
Human stem cell factor-2:470 * 10 -9%-1740 * 10 -9%;
Human stem cell factor: 1.9 * 10 -9%-2.8 * 10 -9%;
Human granulocyte-macrophage colony stimutaing factor: 2.0 * 10 -9%-2.9 * 10 -9%;
CD135 ligandin: 9.4 * 10 -9%-10.8 * 10 -9%;
Human erythropoietin: 1.4 * 10 -9%-2.4 * 10 -9%;
Human epithelial cell generates the factor: 9.4 * 10 -9%-10.8 * 10 -9%;
Human short fibroblast growth factor-b:9.4 * 10 -9%-10.8 * 10 -9%.
2, the nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 according to claim 1 is characterized in that, component and weight percent are:
MEM：59.36％-61％；
RPMI-1640：29.7％-30.14％；
Hanks：9.3％-10.5％；
Human stem cell factor-2:850 * 10 -9%-1260 * 10 -9%;
Human stem cell factor: 2.1 * 10 -9%-2.5 * 10 -9%;
Human granulocyte-macrophage colony stimutaing factor: 2.2 * 10 -9%-2.6 * 10 -9%;
CD135 ligandin: 9.8 * 10 -9%-10.4 * 10 -9%;
Human erythropoietin: 1.7 * 10 -9%-2.1 * 10 -9%;
Human epithelial cell generates the factor: 9.7 * 10 -9%-10.4 * 10 -9%;
Human short fibroblast growth factor-b:9.6 * 10 -9%-10.4 * 10 -9%.
3, a kind of preparation method of nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 as claimed in claim 1 is characterized in that, may further comprise the steps:
1. after getting MEM, RPMI 1640, Hanks mixing in described ratio, fully stir, make basic culture solution;
2. get again in described ratio that human stem cell factor-2, human stem cell factor, human granulocyte-macrophage colony stimutaing factor, CD135 ligandin, human erythropoietin, human epithelial cell generate the factor, the human fibroblast generates the factor-b and adds successively in the basic culture solution, stir, dissolve;
3. with the fetal liver mesenchyma cellular segregation,, get 10 according to the reading of counter with the mesenchymal cell that is purified into 7Individual fetal liver mesenchyma cell is inserted culturing bottle, adds DMEM nutrient solution and 2% human serum and cultivates, and then supernatant liquor is extracted out, wash,
Further add DMEM nutrient solution and 5% human serum and mix in culturing bottle with the fetal liver mesenchyma cell, cultured continuously is all collected supernatant liquor,
Supernatant liquor is through after filtering, and passes through centrifugally again, and the material of whole cell debriss and non-solubility is removed, and makes conditioned medium;
4. with conditioned medium with after MEM mixes, stir the back and form mixed-culture medium, utilize pH regulator to 7.40～7.45 of sodium bicarbonate with mixed-culture medium, can make stem cell CD 34 and strengthen and express nutrient solution through filtering.
4, the preparation method of the nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 according to claim 3 is characterized in that, the tire liver of step described in 3. is meant: get the human tire liver after the 19-20 week standard test.
5, the preparation method of the nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 according to claim 3 is characterized in that, 2% human serum of step described in 3. cultivated, and is meant: the cultivation through 10-14 days,
6, the preparation method of the nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 according to claim 3 is characterized in that, after step supernatant liquor is extracted out described in 3., with Hanks the fetal liver mesenchyma cell in the culturing bottle is washed secondary.
7, the preparation method of the nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 according to claim 3 is characterized in that, before step supernatant liquor is all collected described in 3., through 48 hours cultured continuously, the control of cultured continuously condition required: 37 ℃ of temperature, CO 2Concentration 5%.
8, the preparation method of the nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 according to claim 3 is characterized in that, described filtration is meant: after supernatant liquor filters through 0.2 micron filter.
9, the preparation method of the nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 according to claim 3 is characterized in that, step centrifugal described in 3. is meant: through the high speed centrifugation of 2500g.
10, the preparation method of the nutrient solution of enhancing expression of fetal liver hemopoietic stem cell CD 34 according to claim 3 is characterized in that, the sodium bicarbonate of step described in 4. is meant: 0.1 μ mol NaHCO 3
CNA200810040136XA 2008-07-03 2008-07-03 Culture solution for reinforcing expression of fetal liver hemopoietic stem cell CD34 and preparation thereof Pending CN101314767A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CNA200810040136XA CN101314767A (en) 2008-07-03 2008-07-03 Culture solution for reinforcing expression of fetal liver hemopoietic stem cell CD34 and preparation thereof
PCT/CN2009/000244 WO2010000125A1 (en) 2008-07-03 2009-03-09 The key technologies of isolation, purification, freeze-drying, anabiosis of fetal liver hemopoietic stem cell and the preparation methods

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNA200810040136XA CN101314767A (en) 2008-07-03 2008-07-03 Culture solution for reinforcing expression of fetal liver hemopoietic stem cell CD34 and preparation thereof

Publications (1)

Publication Number Publication Date
CN101314767A true CN101314767A (en) 2008-12-03

Family

ID=40105915

Family Applications (1)

Application Number Title Priority Date Filing Date
CNA200810040136XA Pending CN101314767A (en) 2008-07-03 2008-07-03 Culture solution for reinforcing expression of fetal liver hemopoietic stem cell CD34 and preparation thereof

Country Status (2)

Country Link
CN (1) CN101314767A (en)
WO (1) WO2010000125A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010000125A1 (en) * 2008-07-03 2010-01-07 上海天生生物科技有限公司 The key technologies of isolation, purification, freeze-drying, anabiosis of fetal liver hemopoietic stem cell and the preparation methods

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0329449D0 (en) * 2003-12-19 2004-01-28 Omnicyte Ltd Stem cells
CN1635113A (en) * 2003-12-31 2005-07-06 中山大学 Process for preparing hemapoietic stem cells from mesenchymal stem cells
CN101314767A (en) * 2008-07-03 2008-12-03 上海天生生物科技有限公司 Culture solution for reinforcing expression of fetal liver hemopoietic stem cell CD34 and preparation thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010000125A1 (en) * 2008-07-03 2010-01-07 上海天生生物科技有限公司 The key technologies of isolation, purification, freeze-drying, anabiosis of fetal liver hemopoietic stem cell and the preparation methods

Also Published As

Publication number Publication date
WO2010000125A1 (en) 2010-01-07
WO2010000125A9 (en) 2010-04-01

Similar Documents

Publication Publication Date Title
dos Santos et al. Bioreactor design for clinical‐grade expansion of stem cells
CN101914490B (en) Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof
CN106479971A (en) A kind of serum-free medium for cultivating mescenchymal stem cell and method
CN102191218B (en) Complete medium and human amnion-derived mesenchymal stem cell culture method
CN107475190B (en) Method for clinical-level efficient preparation and cryopreservation of fat SVF cells and application thereof
CN104204192A (en) Mesenchymal stem cells conditioned medium and methods of generating and using the same
CN102660501A (en) Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord
CN1389565A (en) Culture process of human nerve stem cell
CN105238749B (en) A kind of method of recovery mesenchymal stem cell
CN105970300A (en) Method for establishing human umbilical cord mesenchymal stem cell bank by adopting blood serum substituent
CN104651305A (en) Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells
CN102517251A (en) Mesenchymal stem cells, as well as preparation method and application thereof
Lavrentieva et al. Limited potential or unfavorable manipulations? Strategies toward efficient mesenchymal stem/stromal cell applications
CN101831403B (en) Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro
CN106318906A (en) Method for large-scale culture of human umbilical cord mesenchymal stem cells
CN103087977A (en) Culture solution for in vitro efficient amplification of animal cells and application of culture solution
CN108504628A (en) The cultural method of human umbilical cord mesenchymal stem cells
CN102191229A (en) Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC)
Thilly et al. [15] Microcarrier culture: A homogeneous environment for studies of cellular biochemistry
CN104630135B (en) Method for preparing hepatic stem cells on large scale and application thereof
CN106085957B (en) A kind of three-dimensional amplification of microcarrier and the method for activating NK
CN105018422B (en) A kind of method of human umbilical cord mesenchymal stem cells prepare with scale
CN101314767A (en) Culture solution for reinforcing expression of fetal liver hemopoietic stem cell CD34 and preparation thereof
CN102250841A (en) Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof
CN109439617A (en) A kind of stem cell serum-free culture medium and preparation method thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication

Open date: 20081203