WO2006081722A1 - Procédé d’épuration et de culture de cellule souche pluripotente d’éponge de mer - Google Patents

Procédé d’épuration et de culture de cellule souche pluripotente d’éponge de mer Download PDF

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WO2006081722A1
WO2006081722A1 PCT/CN2005/001540 CN2005001540W WO2006081722A1 WO 2006081722 A1 WO2006081722 A1 WO 2006081722A1 CN 2005001540 W CN2005001540 W CN 2005001540W WO 2006081722 A1 WO2006081722 A1 WO 2006081722A1
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cells
sponge
cell
culture
seawater
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PCT/CN2005/001540
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French (fr)
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Wei Zhang
Liming Sun
Meifang Jin
Xingju Yu
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Dilian Institute Of Chemical Physics, Chinese Academy Of Sciences
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Priority to EP05795556A priority Critical patent/EP1911835A4/en
Publication of WO2006081722A1 publication Critical patent/WO2006081722A1/zh

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    • 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
    • C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones

Definitions

  • the invention belongs to the field of marine biological technology, and relates to marine animal cell culture, marine drug production technology and the like. Specifically, it is a method for purifying and culturing marine sponge pluripotent stem cells, and the present invention can be applied to the in vitro proliferation of marine sponge cells and the biological production of sponge bioactive substances and biosilicon materials. Background technique
  • Sponges are low-level marine multicellular animals that feed on filtered seawater.
  • a large number of active and highly active anti-tumor, anti-infective and anti-HIV active substances have been isolated and extracted from the sponge tissue.
  • sponge is by far the largest source of marine natural medicines. Statistics from 2000 years ago show that new compounds found in sponges account for 40% of the total, from the last three years (2002-2004) published in various journals of marine natural In terms of products, the sponges accounted for 36.3 %, 38.2% and 32.6%, respectively. More than 10% of sponge secondary metabolites are cytotoxic, 2% of compounds with cytotoxic activity in other marine organisms, and less than 1% in terrestrial plants and microorganisms.
  • the porous animal such as sponge has certain characteristics in its biological structure, that is, the sponge has a rich water channel system, especially some intertidal sponges with small body (such as the luxuriant membrane sponge).
  • the special porous structure of the sponge animal can not be located in the in vitro culture process. Therefore, in the primary culture, the sponge tissue is generally dispersed, and the mixed sponge cells are isolated, directly cultured or after preliminary separation. Cultivate again.
  • the sponge tissue is generally composed of a plurality of cells such as a progenitor cell, a collagen cell, an osteoblast, a squamous cell, a collar cell, a muscle cell, a pore cell, a gray cell, a small cell, a vesicle cell, a bacterial cell, and the like, wherein the original cell is
  • the pluripotent cells in the sponge tissue have strong proliferative ability and can differentiate into collagen cells, osteoblasts, epithelial cells, and collar cells, which constitute the main cell types of the corpus cavernosum, and the proliferation and differentiation of these cells
  • the object of the present invention is to provide a method for purifying and culturing a sponge pluripotent stem cell (progenitor cell), which can be passed through a sponge original cell in vitro. Large-scale cultivation to produce active substances.
  • the pretreated sponge cells were subjected to differential centrifugation, differential aggregation, differential adhesion and density gradient centrifugation, and separated and purified according to the size and adhesion characteristics of the sponge cells;
  • the mixed sponge cells obtained in step 1 were adjusted for cell density by CMFSW, centrifuged at 300-600 rpm for 10-20 min, the supernatant was discarded, and the cell pellet was retained to obtain a large volume of sponge cells including the original cells. , to be separated next;
  • the cells obtained in step 2.1 were precipitated, washed with CMFSW to remove residual ethylenediaminetetraacetic acid, adjusted to a cell density of 5-10 x 10 7 /ml, and cultured in a container at CMFSW for 8 to 12 hours at 18 to 25 ° C.
  • the protocells are fully involved in aggregation, forming cell aggregates, and then discarding free sponge cells that are not involved in aggregation, and the remaining cells are used for the next separation;
  • the cell aggregates were continuously cultured with artificial seawater to adhere to the cells, and the cells were aggregated.
  • the collagen cells and epithelial cells of ⁇ were completely attached to the bottom of the cell aggregates, and actively migrated and spread: & 70-80% confluence
  • CMFSW containing 2-10 mM ethylenediaminetetraacetic acid
  • the lens is controlled under the microscope at 18-22 ° C.
  • the time of action of diamine tetraacetic acid is 5 ⁇ 10min; the primary cells are initially separated, that is, the collagen cells and the original cells are initially separated, and the next step is to be separated;
  • the primary cells isolated in step 2.3 are adjusted to have a density of 5 ⁇ 10 ⁇ 10 7 /ml, and used; 2.4.2 to prepare a gradient liquid;
  • each layer of the gradient solution is l ⁇ 10ml, the sample volume is l ⁇ 5ml, and the total cell number is 2 ⁇ 20> ⁇ 10 7 ; the collected original cells are washed away with the CMFSW to the density gradient medium, and the horizontal centrifuge is used. 500-1500 rpm, 5 ⁇ 20min, washing 2 ⁇ 4 times, further separating the original cells, ie pluripotent stem cells, from other cells.
  • step 2.3 it is preferred to discriminate the original cells distributed in the upper layer of the cell aggregate with CMFSW containing 2 to 5 mM ethylenediaminetetraacetic acid;
  • Step 2.4 The preparation of the gradient liquid may also be: using a sucrose density gradient centrifugation, the sucrose concentration gradient is 12-24%, 24-36%, 36-48%, 48-52%, 58-60%; The density-gradient centrifugation of the primary discrete cells after adjusting the density is carried out.
  • each layer of the gradient solution is 1 to 2 ml, the sample loading is 1-2 ml, and the total cell number is 2 to 5 ⁇ 10 7 ; or 50 ml is used.
  • each layer of gradient liquid is 5 ⁇ 10ml, the loading amount is 3 ⁇ 5ml, and the total cell number is 1 ⁇ 2 ⁇ 10 8 ;
  • the in vitro proliferation culture and metabolic examination steps of the sponge cells can be added, specifically: the pluripotent stem cells isolated in the step 1 are used for culture; the cell seeding density is 2 ⁇ 10 6 ⁇ 3 ⁇ 10 7 cells/ml, every 1 ⁇ 2 days during the culture process.
  • the composition of the artificial seawater culture medium is improved: the seawater basal medium is added with a sponge interstitial fluid extract, other growth factors or differentiation control factors; and the controlled culture can be irradiated or darkly cultured according to the sponge type;
  • Preparation of the sponge interstitial fluid extract Take a freshly collected healthy sponge block and soak it in CMFSW at a weight/volume ratio of 1/10 ⁇ 1/20, and replace 80 ⁇ 100% of CMFSW for an average of 2-4 hours. 4 ⁇ 6 times, collect all the leachate, sterile filtration, obtain the leachate for sponge cell culture; mix with the sponge cell basal medium according to the ratio of 25% ⁇ 75% of the leachate;
  • the growth factor is: 5-20 mg/ml soluble cholesterol, 5-20 ⁇ ⁇ / ⁇ 1 hydrocortisone, 5-10 ⁇ ⁇ / ⁇ 1 Insulin, 5-l ( ⁇ g/ml transferrin, 6 10xl (T 5 g/L Na 2 Se0 3 , 15-50 ⁇ ⁇ / ⁇ 1 phytohemagglutinin (PHA), lOO OO g/ml dithiothreose) Alcohol, one of 2-8 x 10 4 g / L glutathione or a combination thereof;
  • the differentiation control factor is: 5-50 ⁇ M/ml hydroxyurea, one of 5-20 u M/ml retinoic acid or a combination thereof.
  • the present invention is coupled to obtain purified sponge pluripotent stem cells by a plurality of separation methods as seed cells for further cultivation.
  • the difference from conventional enrichment of spongy cells by density gradient centrifugation is that the present invention maximizes the purification of protocells by differential centrifugation, cell-specific aggregation, and differential adhesion prior to density gradient centrifugation.
  • the interference of other kinds of sponge cells on density gradient centrifugation not only improves the purity of the isolated original cells (the purity of the original cells is greater than 80%), but also improves the recovery rate of the original cells (the recovery rate of the original cells is 50-60%) Moreover, the viability of the original cells obtained after separation is above 95%, and the high-efficiency separation and purification of the sponge cells is facilitated, which is beneficial to further study the biological properties of the sponge stem cells and the in vitro culture rules.
  • the pluripotent stem cell culture step isolated and purified according to the present invention can promote the proliferation of the original cells and inhibit the differentiation of the original cells by adding growth factors and differentiation control factors.
  • the sponge protocell isolated and purified by the present invention can be subjected to large-scale culture in vitro to carry out biosynthesis of the active substance.
  • Figure 1-1 is a diagram showing the composition of sponge cells before differential centrifugation according to the present invention (mixed sponge cells ( ⁇ 400)).
  • FIG 1-2 shows the immunohistochemical staining of 5-bromo-2-deoxyuracil (BrdU) before differential centrifugation according to the present invention (mixed sponge cells BrdU immunohistochemical staining ( ⁇ 400)).
  • Figure 1-3 is a diagram showing the immunohistochemical staining of PCNA before differential centrifugation according to the present invention (mixed sponge cell PCNA immunohistochemical staining (x400)).
  • Figure 1-4 is a schematic diagram of the composition of the sponge cells after differential centrifugation according to the present invention (500 rpm, sponge cells after centrifugation at 15 min.
  • Figure 1-5 shows the immunohistochemical staining of BrdU after differential centrifugation in the present invention (500 rpm, sponge cells after centrifugation for 15 min via BrdU) Immunohistochemical staining (x400)).
  • Figure 1-6 shows the immunohistochemical staining of PCNA after differential centrifugation according to the present invention (500 rpm, PCNA immunohistochemical staining of sponge cells after 15 min centrifugation ( ⁇ 400)).
  • Figure 2-1 shows the differential aggregation of sponge cells of the present invention.
  • GOmin rapidly aggregates cells to form cell clusters ( ⁇ 40)).
  • Fig. 2-2 is a differential aggregation diagram of sponge cells of the present invention (cultured for 2 hours, cell clusters began to adhere, and collagen cells began to migrate outward (x40)).
  • Fig. 2-3 is a differential aggregation diagram of sponge cells of the present invention (cultured for 4 hours, collagen cells continue to migrate, and cell clusters begin to spread (x40)).
  • Figure 2-4 is a differential aggregation diagram of sponge cells of the present invention (cultured for 6 hours, cell clusters continue to spread, and confluence begins with collagen cells (x250)).
  • 2-5 are differential aggregation diagrams of sponge cells of the present invention (8 hours of culture, maximum fusion 040 between cell clusters)).
  • Fig. 2-6 is a differential aggregation diagram of the sponge cells of the present invention (the original cells are dissociated from the collagen cells, and the collagen cells are still in an adherent state (x400)).
  • Fig. 2-7 is a differential aggregation diagram of sponge cells of the present invention (sparsely aggregated sponge cells mixed with a part of collagen cells and a small number of small cells (x400)).
  • Fig. 2-8 is a differential aggregation map of sponge cells of the present invention (BrdU immunohistochemical staining of differentially aggregated cells ( ⁇ 400)).
  • Fig. 2-9 is a differential aggregation map of sponge cells of the present invention (PCNA immunohistochemical staining of differentially aggregated cells ( ⁇ 400)).
  • Fig. 2-10 is a diagram showing the process of differential adhesion adherence of sponge cells of the present invention (dissociated cells after differential adhesion ( ⁇ 400)) ⁇
  • Fig. 2-11 is a diagram showing the process of differential adhesion and adherence of sponge cells of the present invention (BrdU immunohistochemical staining (x400) of cells dissociated after differential adhesion).
  • Fig. 2-12 is a diagram showing the process of differential adhesion adherence of sponge cells of the present invention (PCNA immunohistochemical staining (x400) of sponge cells dissociated after differential adhesion).
  • Figure 3-1 is a schematic diagram of the density gradient centrifugation purification of spongy cells of the present invention (density gradient layer prepared by the combination of Ficoll and diatrizoate).
  • Figure 3-2 is a schematic view showing the distribution of spongy cells after density gradient centrifugation according to the present invention.
  • Figure 4-1 is a schematic diagram of the density gradient centrifugation of the present invention (C5 layer of protocells ( ⁇ 400)).
  • Fig. 4-2 is a diagram showing the immunohistochemical staining of BrdU after isolation and purification according to the present invention (BrdU. positive cells ( ⁇ 400) in the original cells).
  • Fig. 4-3 is a diagram showing the immunohistochemical staining of PCNA after isolation and purification according to the present invention (protocell PCNA positive cells ( ⁇ 400)).
  • Figure 5 is a graph showing the percentage of spongy cells obtained by the steps of the mixed sponge cells of the present invention and BrdU and
  • PCNA Percentage of PCNA (MC is mixed cells; DC is differential centrifugation; DA is differential aggregation; DAD is differential adhesion; DGC is density gradient centrifugation).
  • Figure 6 is a graphical representation of the effect of different incubation times on the uptake of BrdU by protocells in the present invention.
  • Fig. 8 is a growth kinetic curve of the in vitro culture of the sponge mixed cells and the purified original cells of the present invention (the cell growth is expressed by the number of cells).
  • Fig. 9-1 is a view showing the morphology of cells obtained in each step of the separation process of the sponge cells of the South China Sea (prepared mixed cells).
  • Fig. 9-2 is a view showing the morphology of cells obtained by the steps of the separation process of the sponge cells of the South China Sea in the present invention (slow-speed centrifugation, sponge cells obtained after low-speed centrifugation at 500 rpm, lOmin).
  • Fig. 9-3 is a diagram showing the morphology of cells obtained by the steps of the separation process of the sponge cells of the South China Sea (cell aggregates formed after differential aggregation).
  • Fig. 9-4 is a view showing the morphology of cells obtained by the steps of the separation process of the sponge cells of the South China Sea (the original cells are dissociated from the epithelial cell layer adhered to the bottom of the culture flask).
  • Fig. 9-5 is a view showing the morphology of cells obtained by the steps of the separation process of the sponge cells of the South China Sea (an unknown type of cortical cells separated and purified by density gradient centrifugation).
  • Fig. 9-6 is a view showing the morphology of cells obtained by the steps of the separation process of the sponge cells of the South China Sea (the original cells separated and separated by density gradient centrifugation). .
  • Figure 10-1 shows the distribution of cells after sedimentation at 10% sucrose rate in the separation of sponge cells from the South China Sea.
  • Figure 10-2 shows the distribution of cells after centrifugation by sucrose density gradient when separating the sponge cells from the South China Sea.
  • Fig. 10-3 is a diagram showing the morphology of cells obtained by the steps of the separation process of the sponge cells of the South China Sea (the bottom cells obtained after 10% sucrose sedimentation).
  • Figure 10-4 shows the in vitro culture growth curve of the sponge mixed cells and purified protocells of the South China Sea.
  • Fig. 11 is a view showing the cell growth of the mixed cells in vitro cultured by the MTT method in the comparative example of the present invention.
  • Fig. 12-1 is a photograph showing the mold contamination occurring in the in vitro culture of the mixed cells of the comparative example of the present invention (the fungal hyphae are entangled between the sponge cell clusters ⁇ )
  • Fig. 12-2 is a photograph showing the bacterial contamination occurring in the in vitro culture of the mixed cells of the comparative example of the present invention (in which the bacteria and protozoa absorb the sputum and become blue-purple, and the cells are still brownish yellow). detailed description ,
  • the Chinese Huang Bohai microscopic membrane sponge was taken, and after being pretreated, the sponge pluripotent stem cells (ie, the original cells) were effectively separated and highly purified by using four-step coupling separation, and then the original cells were cultured and examined.
  • CMFSW sterile seawater containing gentamicin (400ug/ml)
  • CMFSW sterile seawater containing gentamicin (400ug/ml)
  • CMFSW sterile seawater containing gentamicin (400ug/ml)
  • the mixed sponge cells remaining in step 1 were adjusted to a cell density of 5 ⁇ 10 7 /ml with CMFSW, and then centrifuged at a small rotation speed, 300 rpm, 15 min, the supernatant was discarded, and the cell pellet was retained to obtain a cell including the original cells. Larger volume of sponge cells (see Figure 1-4); Count the percentage of protocells, and select some cells to investigate the percentage of positive cells in PCNA (see Figure 1-6) and BrdU (see Figure 1-5). Separate in the next step.
  • the cells obtained in the step 2.1 were precipitated, washed twice with CMFSW to remove residual EDTA, adjusted to a cell density of 10 ⁇ 10 7 /ml, and cultured in a 100 ml volume glass culture flask, and the actual culture volume was 10 ml/bottle, 18 ° C, 8 Hour, shake culture, 20 rpm, so that the original cells fully participate in aggregation, form cell aggregates (see Figure 2-1), discard the free sponge cells that are not involved in aggregation, and collect some of the cell samples to count the cell aggregates.
  • the percentage of protocells see Figure 2-7) and the positive rate of PCNA (see Figure 2-9) and BrdU (see Figure 2-8) were detected, and the remaining cells were used for the next step.
  • ASW artificial sea water, artificial seawater containing 460 mM NaCl, 7 mM Na 2 S0 4 , 10 mM KC1, 10 mM CaCl 2 , 50 mM MgCl 2 , 10 mM Hepes, ⁇ ⁇ . ⁇ ).
  • the discrete protocell aggregates are fully dispersed and blown (see Figures 2 ⁇ 10).
  • the primary cells are initially separated, and the epithelial/collagen cells and the original cells are initially separated.
  • the percentage of protocells in the sample is counted and examined.
  • PCNA see Figure 2-12
  • BrdU see Figure 2-11
  • Proliferating cell nuclear antigen was used to investigate the proliferative capacity of spongellocytes.
  • the experimental data showed that as the separation process progressed, the proportion of protocells in the isolated cells gradually increased, and the proportion of PCNA-positive cells increased.
  • Figure 5 shows that the isolated cells have a strong proliferative capacity, thereby demonstrating that the isolated cells are spongy cells.
  • the light source is a fluorescent lamp.
  • the artificial seawater culture medium is improved: a sponge interstitial fluid extract is added to the seawater basic medium.
  • the basic medium for seawater is: 460 mM NaCl (sodium chloride), 7 mM Na 2 S0 4 (sodium sulfate), 10 mM KCl (potassium chloride), 10 mM CaCl 2 (calcium chloride), 50 mM MgCl 2 (magnesium chloride), 20 mM Tris (trihydroxycarbamidine), 30 mM Na2SiO3.9H2O (sodium silicate), 60 ⁇ FeC 6 H 5 0 7 .5H 2 0 (iron citrate), 4 ⁇ ZnCl 2 (zinc chloride), 5 ⁇ Vit C (vitamin C), 10 ⁇ L-Glutamine (glutamine), 10 ⁇ L-asparagin (asparagine), ⁇ glycine (glycine), 20 ⁇ glycose (glucose), ImM sodium pyruvate (
  • Spongy cells were prepared according to step 1, and the above-mentioned modified seawater medium was used for in vitro culture to examine cell proliferation and metabolic dynamics, and compared with sponge mixed cell culture.
  • Sponge cells and mixed sponges The proliferation of cells in vitro is shown in Figure 7 and Figure 8.
  • the number of cells and the expression of metabolites were examined.
  • the synthesis of ⁇ -carotene can reach 0.01 mg/(10 6 cells/ml) ( , ' 5a-c olesterol-3 ⁇ -ol can reach 0.1m g /5xl0 6 cells/ml.
  • ⁇ The active substance can also be produced by large-scale in vitro culture of spongy cells by the present invention.
  • the principle and method of separation from the sponge cells of the Yellow Sea are basically the same, but due to the difference in sedimentation coefficient and aggregation adhesion between different species of sponge cells, the above method can be slightly improved to obtain the stem cells of the South Sea sponge. . the difference lies in:
  • Step 1 The Nanhai healthy sponge tissue block that was kept for 14 days in the aquarium tank was washed with natural seawater containing gentamicin (800 ug/ml) to remove surface microorganisms and other commensal organisms, and then cut into 2 to 3 mm 3 small pieces. Then, CMFSW (986 mM NaCl, other components as above) was washed 4 times for 3 hours each time, and CMFSW containing EDTA was added and shaken, and then filtered, centrifuged at 2000 rpm for 10 minutes, and the cells were resuspended with CMFSW containing 2 mM EDTA to obtain mixed sponge cells. .
  • CMFSW 986 mM NaCl, other components as above
  • Step 2.1 Take step 1 to mix the sponge cells and adjust the cell density to 2 ⁇ 10 7 /ml with CMFSW to prepare mixed cells (see Figure 9-1). After mixing the sponge cells at 500 rpm for 10 minutes, enrich the volume. Large cortical and medullary sponge cells (see Figure 9-2).
  • Step 2.2 The cells obtained in step 2.1 were precipitated, washed with CMFSW, adjusted to a cell density of 5> ⁇ 10 7 /ml, cultured in a glass culture flask, 25 V, 12 hours, shaken, and discarded free sponge not involved in aggregation. Cells, cell aggregates were used for the next step of separation (see Figure 9-3).
  • Step 2.3 Switch to ASW (986mM NaCl, other components as above) and continue to culture the cell aggregates for 10 hours to adhere to the wall.
  • the collagen cells and epithelial cells in the aggregates are well attached to the bottom of the cell aggregates and When the active migration and spreading reached 70% confluence, the culture was stopped.
  • CMFSW containing 5 mM EDTA (see Figure 9-4), and the EDTA was controlled under the microscope.
  • the time of action, at 18 ° C, the treatment time is l Omin (if using 10 mM EDTA, 5miri is treated).
  • the discrete protocell aggregates are fully dispersed and uniformly blown, and the primary cells which are initially separated are initially separated from the other cells;
  • Step 2.4
  • the sponge cells isolated in step 2.1 were first subjected to a speed sedimentation at a low concentration of sucrose (10% in this example), and the cells were divided into three parts according to the sedimentation rate (see Fig. 10-1), wherein the uppermost layer was volume-compared.
  • Small cortical 'layer cells the second layer is the larger cortical and medullary layer cells and a small number of original cells, accounting for about 10%;
  • the third layer that is, the sedimentation rate, the largest one is the larger non-original A mixture of cells and protocells, of which about 65% of the original cells; take the fastest sedimentation rate of cells in step 2.2 (see Figure 10-3) and continue the sucrose density gradient centrifugation with a sucrose concentration gradient of 20%, 30%.
  • the gradient solution is 8 ml per layer, the loading amount is 5 ml, and the total number of cells is lx lO 8 ; the collected original cells are washed with CMFSW to remove the density gradient medium, and the horizontal centrifuge is used. , 1500 rpm, lOmin, washed a total of 3 times, further separating the original cells, ie, pluripotent stem cells, from other cells.
  • Step 3 The stem cells isolated in step 1 are cultured; the cell seeding density is 2 ⁇ 10 6 C eUs/ml, and 70% of the modified seawater medium is replaced every other day during the culture, and the culture temperature is controlled at 25 ° C (dark culture).
  • the culture was shaken at 40 rpm; the number of cells was counted, and the cell proliferation was compared with the sponge-mixed cell culture; the modified seawater medium was added to the seawater basal medium as: 60 g/L Na 2 Se0 3 , lmg/L dithiothreitol (DTT) and 240 g/L glutathione (GSH).
  • DTT dithiothreitol
  • GSH glutathione
  • Embodiment 2 The difference from Embodiment 2 is that:
  • Step 1 The Nanhai healthy sponge tissue block, which was kept for 7 days in the aquarium tank, was washed with natural seawater containing gentamicin (200 ug/ml) to remove surface microorganisms and other commensal organisms, and then cut into l ⁇ 3 mm 3 small pieces. After washing with CMFSW (986 mM NaCl, other components as above) for 5 times, 2.5 hours each time, adding CMFSW containing EDTA', shaking and discarding, filtering, 2500 rpm, 20 min, resuspending the cells with CMFSW containing 3 mM EDTA to obtain a mixture. Sponge cells.
  • CMFSW 986 mM NaCl, other components as above
  • Step 1 Take step 1 Mix the sponge cells and adjust the density of the cells with CMFSW to prepare mixed cells. After mixing the sponge cells at 600 rpm for 20 min, enrich the cortical layer and medullary layer sponge cells.
  • Step 2.2 The cells obtained in the step 2.1 were precipitated, washed with CMFSW, adjusted to a cell density of 7 ⁇ 10 7 /ml, cultured in a glass culture flask, and shaken at 20 ° C for 10 hours to discard the free sponge cells not involved in aggregation. Cell aggregates were used for the next step of separation.
  • Step 2.3 Switch to ASW (986mM NaCl, other components as above) and continue to culture the cell aggregates for 10 hours to adhere to the wall.
  • the collagen cells and epithelial cells in the aggregates are well attached to the bottom of the cell aggregates and When the active migration and spreading reached 75% confluence, the culture was stopped.
  • CMFSW containing 6 mM EDTA, and the time of EDTA control was controlled by microscope, 20 ° C.
  • the treatment time was 5 min ; if 5 mM EDTA was used, it was treated for 7 min.
  • the discrete protocell aggregates are fully dispersed and uniformly blown, and the primary cells which are initially separated are initially separated from the collagen cells;
  • Step 2.4
  • the sponge cells isolated in step 2.1 are first subjected to a speed sedimentation at a low concentration of sucrose (20% in this example), followed by sucrose density gradient centrifugation, and the sucrose gradient is 12%, 24%, 36%, 48%, 60. %. 3)
  • the gradient liquid of each layer is 10ml
  • the sample volume is 3ml
  • the total cell number is 2> 10 8 ;
  • the collected original cells are washed away with the CMFSW to the density gradient medium, using a horizontal centrifuge, 500rpm, 15 min, a total of 4 washes, further separating the original cells from other cells.
  • Step 3 Control the culture temperature at 25 ° C (dark culture, the South China Sea sponge is generally dark culture, because it grows deeper sea water);
  • the improved sponge cell basal medium component can be added to the seawater basal medium as: 20 ⁇ M/ml hydroxyurea and 10 ⁇ M/ml retinoic acid.
  • the original cells isolated in step 1 were cultured; the cell seeding density was 3 ⁇ 10 7 C ells/ml, and 70% of the modified seawater medium was replaced every other day during the culture, and the culture temperature was controlled at 25 ° C and 20 rpm. Culture; Count the number of cells, compare cell proliferation with sponge-mixed cell culture.
  • the invention is based on the theory that the sponge primitive cells are pluripotent stem cells in the sponge tissue and has strong proliferation ability.
  • the technical route of in vitro culture of the sponge stem cells is first proposed, and the four-step coupling separation method of the invention realizes the original cells of the sponge. Effective separation and high purification.
  • the process conditions of in vitro culture of sponge stem cells were further established, including the physicochemical conditions of culture, the medium, the key factors regulating differentiation and growth, the culture method and the core technology of cultured bioreactor. In vitro culture and stable proliferation of sponge cells are achieved, and sponge bioactive substances can be efficiently produced in the sponge system tested.
  • the invention provides a new technical way for solving the production of important drug source organisms and sponge bioactive substances, and has great significance for the industrialization of marine drugs, especially sponge drugs, and also for the cell biology of marine invertebrates. Research provides important means and has important theoretical significance and application value. The expected business prospects will be extremely broad.
  • the improved sponge cell basal medium component can also add a growth factor to the artificial seawater culture medium: one of soluble cholesterol, hydrocortisone, insulin, transferrin, phytohemagglutinin (PHA) or a combination thereof. Or a differentiation control factor of one of retinoic acid and hydroxyurea.
  • a growth factor to the artificial seawater culture medium: one of soluble cholesterol, hydrocortisone, insulin, transferrin, phytohemagglutinin (PHA) or a combination thereof.
  • PHA phytohemagglutinin
  • the mixed cells obtained by the conventional chemical dispersion method are directly agglomerated in ASW or natural seawater.
  • 100 U/ml gentamicin or penicillin 100 U/ml, streptomycin 100 g/ml, Amphotericin 3 g/ml was cultured, and the growth of the cells was measured by MTT method.
  • the results are shown in Fig. 11.
  • the cell culture system began to increase the MTT detection value from the fourth day, and reached the initial MTT value on the seventh day. More than double, after microscopic examination, a large number of bacteria and molds (see Figures 12-1, 12-2) were found to grow, and it was concluded that the abnormal growth of the measured biomass was the growth of bacteria and mold.

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Description

海洋海绵多能干细胞的纯化与培养方法 技术领域
本发明属于海洋生物技术领域， 涉及海洋动物细胞培养， 海洋药物生产技术等。 具体地说是一种海洋海绵多能干细胞的纯化与培养方法，本发明可应用于海洋海绵细 胞的体外增殖与海绵生物活性物质及生物硅材料等的生物生产。 背景技术
海绵是低等海洋多细胞动物，靠过滤海水为食。从海绵组织中已分离提取出大量 的结构新颖、 活性极强， 具有抗肿瘤、 抗感染、 抗 HIV的活性物质。 事实上海绵是 迄今为止海洋天然药物的最大来源， 2000年前的统计表明从海绵中发现的新化合物 占总数的 40%, 从最近三年 （2002-2004) 在各类杂志上发表的海洋天然产物来看， 来源于海绵的分别占到了 36.3 %、 38.2%和 32.6%。超过 10%的海绵次生代谢产物有 细胞毒性， 在其它海洋生物中有细胞毒活性的化合物比例为 2%， 而在陆地植物和微 生物中则都小于 1%。 其中 Ara-A和 Avarol已进入临床应用， 有近十种海绵天然产 物正在进行临床前和各期临床研究。 但大多数海绵生物活性物质在海绵体内含量甚 微， 捕捞不能满足药用资源量的需求； 而人工养殖海绵的条件复杂， 且因不能调控高 产，难以实现大规模制备生物活性物质； 同时许多药物分子的复杂结构使得化学合成 不但成本高、合成效率低， 且伴随着严重的化学污染。因此尽管海绵活性物质发现众 多， 真正进入市场的产品极少， 绝大多数海绵药物专利都面临所谓的"程序性药物研 究死亡"的共性问题， 即因缺乏经济可行的生产技术提供充足的药源满足后续临床实 验的要求而停止开发。因此研究和建立海绵生物药源生产的平台技术，实现有潜力开 发成药的海洋天然产物的可控、 高效和规模化生产， 是突破海绵生物药物开发和产 业化瓶颈的关键和核心。通过海绵细胞体外生物反应器培养生产活性产物的研究日渐 引起国际上的广泛关注，并成为解决海绵活性物质供给不足这一瓶颈问题的最有希望 的途径之一。但是实现海绵细胞体外生物反应器大规模培养生产活性物质，还有很多 问题需要解决。首先， 海绵这种多孔动物在其生物学组织结构上有一定的特点， 即海 绵有丰富的水沟系， 尤其是一些体态较小的潮间带海绵 （例如繁茂膜海绵）。 海绵动 物特殊的多孔性结构导致在体外培养过程中无法进行定位取材， 因此，在进行原代培 养时，一般都是将海绵组织整体进行离散， 分离出混合海绵细胞， 直接培养或经初步 分离后再培养。
海绵组织一般由原细胞、胶原细胞、造骨细胞、扁平上皮细胞、领细胞、肌细胞、 孔细胞、 灰细胞、 小球细胞、泡细胞、细菌性细胞等多种细胞构成， 其中原细胞是海 绵组织中的多能千细胞， 具有较强的增殖能力， 并且能够分化成为胶原细胞、造骨细 胞、 上皮细胞、领细胞， 这些构成海绵体的主要细胞类型， 而这些细胞的增殖、 分化
1
确 认 本 能力则丧失或减弱。 ^ 对于重要药源生物-海绵细胞体外培养这一挑战性的课题 选择具有较强增殖能 力的原细胞进行体外培养无论对海绵体外培养细胞系的建立还是进一步反应器放大 培养生产活性代谢产物都是至关重要的，而对该类型海绵细胞的分离纯化则是所有上 述工作的前提。由于对海绵细胞膜表面抗原，尤其是原细胞种类特异性膜蛋白的研究 很少， 加之不同海域、不同种属海绵差异性较大， 目前还没有明确的用于海绵原细胞 鉴别的特异性标志物， 也没有规范的、普适的、特异的原细胞分离方法。 因此， 国内 外的海绵细胞培养工作，大多进行混合海绵细胞培养，原细胞的分离方法也多为密度 梯度离心方法， 难以实现海绵原细胞的高度纯化。 发明内容
为了克服现有技术中大量无增殖能力海绵细胞共同培养的缺陷，本发明的目的在 于提出一种海绵多能干细胞（原细胞）的纯化和培养方法， 采用本发明的同时可以通 过海绵原细胞体外大规模培养来生产活性物质。
为了实现上述目的， 本发明技术方案如下：
将经过预处理的海绵细胞依次进行差速离心、差速聚集、差速黏附和密度梯度离 心， 根据海绵细胞的大小和粘附特性进行分离、 纯化；
1 ) 预处理 （制备混合海绵细胞）
取新鲜或经水族槽暂养 7~14天的健康海绵组织块，用含 200~80(^g/ml庆大霉素 的天然海水洗涤， 剔除杂质； 再将组织块切割成 l~3mm3小块， 浸泡于无钙镁海水 (CMFSW) 中洗涤 3~5次， 每次 2~3小时， 振荡离散出海绵细胞， 过滤， 离心； 再 用含 l~2mM乙二胺四乙酸（Ethlenediaminetetm-acetic acid缩写： EDTA)的 CMFSW 制备细胞悬液，获得混合海绵细胞，待下一步分离；其中离心速度为： 1000~3000rpm， 10~20min;
2 )海绵多能干细胞的纯化
2.1差速离心
将取步骤 1获得的混合海绵细胞用 CMFSW调整细胞密度， 再以 300~600 rpm， 10~20min转速离心， 弃上清液， 保留细胞沉淀， 得到富集包括原细胞在内的大体积 海绵细胞， 待下一步分离；
2.2差速聚集
将步骤 2.1所得细胞沉淀， 用 CMFSW洗涤以除去残存的乙二胺四乙酸， 调整细 胞密度为 5~10xl07/ml，于容器中用 CMFSW在 18~25°C下培养 8~12小时，使所述原 细胞充分参与聚集， 形成细胞聚集体， 然后弃去未参与聚集的游离海绵细胞， 其余细 胞用于下一步分离；
2.3 差速黏附 用人工海水继续培养所述细胞聚集体使之贴壁，待细胞聚集 .Φ的胶原细胞和上 皮细胞完全贴壁分布于细胞聚集体底部， 并进行活跃迁移、 铺展： & 70~80 %汇合时， 停止培养， 再用 CMFSW洗去人工海水后， 用含 2~10mM 乙二胺四乙酸的 CMFSW 将分布于细胞聚集体上层的原细胞离散下来， 在 18~22°C下， 镜下控制乙二胺四乙酸 作用的时间 5~10min; 得初步分离原细胞， 即初步将胶原细胞和原细胞分离， 待下一 步分离；
2.4密度梯度离心
2.4.1将步骤 2.3所得初步分离的原细胞， 调节其密度为 5〜10xl07/ml， 备用； 2.4.2配制梯度液；
2.4.3将调节密度后的初步离散的原细胞进行密度梯度离心
用试管， 每层梯度液为 l~10ml， 上样量为 l~5ml， 总细胞数量为 2~20>< 107 ; 收 集后的原细胞用 CMFSW洗去密度梯度介质，用水平离心机， 500-1500rpm, 5~20min, 共洗涤 2~4次， 从而进一步将原细胞即多能干细胞与其他细胞分开。
另夕卜，所述步骤 2.3中最好用含 2~5mM 乙二胺四乙酸的 CMFSW将分布于细胞 聚集体上层的原细胞离散下来；
步骤 2.4中所述梯度液的配制是： 用 CMFSW将 Ficoll粉末配制成 3~9%Ficoll (p=l .0276-1.0445g/ml, 16~20°C ), 并将 76%的泛影葡胺原液稀释成为 34~38% (p= 1.1379-1.1486g/ml, 16~20°C )，配制密度为 1.04， 1.05 , 1.06， 1.07, 1.08， 1.09, 1.10g/ml 的梯度液； 步骤 2.4中所述梯度液的配制还可以是： 用蔗糖密度梯度离心， 蔗糖浓度 梯度为 12~24%， 24-36% , 36-48% , 48-52% , 58-60%； 步骤 2.4中所述将调节密 度后的初步离散的原细胞进行密度梯度离心， 用 15ml试管时， 每层梯度液为 l~2ml， 上样量为 l~2ml，总细胞数量为 2〜5χ107;或采用 50ml试管时，每层梯度液为 5~10ml， 上样量为 3~5ml， 总细胞数量为 1~2χ108;
本发明可加设海绵细胞的体外增殖培养与代谢考察步骤，具体：采用步骤 1分离 的多能干细胞进行培养； 细胞接种密度 2χ106〜3χ107 cells/ml, 培养过程中每隔 1~2 天更换 50~70%的改进人工海水培养基液， 控制培养温度在 18~25 °C， 在静置或 20〜 40rpm转速下振荡培养； 计数总细胞、 原细胞数量， 检测海绵细胞中增殖细胞核抗原 (PCNA) 阳性率， 并与海绵混和细胞培养进行比较细胞增殖情况； 其中： 所¾！改进 人工海水培养基液成分为：海水基础培养基添加海绵细胞间质液提取物、其它生长因 子或分化控制因子； 所述控制培养可根据海绵种类进行光照或暗培养；
所述海绵细胞间质液提取物制备： 取新采集健康海绵块， 以 1/10~1/20 的重量 / 体积比浸泡于 CMFSW中， 平均 2-4小时更换 80~100%的 CMFSW, 共 4~6次， 收集 全部浸出液， 无菌过滤， 获得用于海绵细胞培养的浸出液； 按照浸出液占 25 %~75 % 的比例与海绵细胞基础培养基混合使用；
所述生长因子为： 5-20mg/ml可溶性胆固醇， 5-20μ§/ηι1氢化可的松， 5-10μ§/ιη1 胰岛素， 5-l(^g/ml转铁蛋白， 6 10xl(T5g/L Na2Se03， 15-50μ§/πι1植物血球凝集素 (PHA), lOO OO g/ml二硫苏糖醇、 2-8xlO_4 g/L谷胱甘肽之一或其组合；
所述分化控制因子为： 5-50 μ M/ml羟基脲、 5-20 u M/ml视黄酸之一或其组合。 与以往的文献和专利比较， 本发明的优点如下：
1 ) 本发明通过多种分离方法耦合获得纯化的海绵多能干细胞， 作为进一步培养 的种子细胞。与常规利用密度梯度离心法富集海绵原细胞的不同之处在于，本发明在 进行密度梯度离心前，采用差速离心、细胞特异性聚集、差速黏附法最大限度地纯化 了原细胞，减少了其他种类海绵细胞对密度梯度离心的干扰，不但提高了分离所得原 细胞的纯度 （原细胞纯度大于 80%)， 还提高了原细胞的回收率 （原细胞的回收率为 50-60%) , 而且分离后所得原细胞的活力在 95 %以上， 实现了海绵原细胞的高效分 离纯化， 有利于下一步深入研究海绵干细胞生物学性质及其体外培养规律。
2)采用本发明分离纯化的多能干细胞培养步骤， 通过添加生长因子和分化控制 因子， 可以促进原细胞增殖、 抑制原细胞分化。
3 )通过本发明所分离纯化出的海绵原细胞进行体外大规模培养， 可以进行活性 物质的生物合成。 附图说明
图 1-1为本发明差速离心前海绵细胞构成图 （混合海绵细胞 （χ400) )。
图 1-2为本发明差速离心前 5-溴 -2脱氧尿嘧啶（BrdU)免疫组织化学染色图（混 合海绵细胞 BrdU免疫组织化学染色（χ400))。
图 1-3为本发明差速离心前 PCNA免疫组织化学染色图 （混合海绵细胞 PCNA 免疫组织化学染色（x400))。
图 1-4 为本发明差速离心后海绵细胞构成图 （500rpm， 15min离心后海绵细胞 图 1-5为本发明差速离心后 BrdU免疫组织化学染色图 （500rpm， 15min离心后 海绵细胞经 BrdU免疫组织化学染色 （x400))。
图 1-6为本发明差速离心后 PCNA免疫组织化学染色图（500rpm， 15min离心后 海绵细胞 PCNA免疫组织化学染色 （χ400))。
图 2-1为本发明海绵细胞的差速聚集图 GOmin快聚集细胞形成细胞团 （χ40))。 图 2-2为本发明海绵细胞的差速聚集图（培养 2小时， 细胞团开始贴壁， 胶原细 胞开始向外迁移 （x40))。
图 2-3为本发明海绵细胞的差速聚集图（培养 4小时， 胶原细胞继续迁移， 细胞 团开始铺展 （x40))。
图 2-4为本发明海绵细胞的差速聚集图 （培养 6小时， 细胞团继续铺展， 并通过 胶原细胞开始汇合（x250))。 图 2-5为本发明海绵细胞的差速聚集图 （培养 8小时， 细胞团间达到最大融合 040))。
图 2-6为本发明海绵细胞的差速聚集图（原细胞从胶原细胞上解离下来， 胶原细 胞仍处于贴壁状态（x400))。
图 2-7为本发明海绵细胞的差速聚集图（差速聚集的海绵细胞， 混有部分胶原细 胞和少量小细胞 （x400))。
图 2-8为本发明海绵细胞的差速聚集图 （差速聚集细胞的 BrdU免疫组织化学染 色（χ400))。
图 2-9为本发明海绵细胞的差速聚集图（差速聚集细胞的 PCNA免疫组织化学染 色 （χ400))。
图 2-10为本发明海绵细胞的差速黏附贴壁过程图 （差速黏附后， 解离下来的细 胞 ( χ400 ) ) ο
图 2-11为本发明海绵细胞的差速黏附贴壁过程图 （差速黏附后， 解离下来细胞 的 BrdU免疫组织化学染色（ x400 ) )。
图 2-12为本发明海绵细胞的差速黏附贴壁过程图 （差速黏附后， 解离下来海绵 细胞的 PCNA免疫组织化学染色 （x400))。
图 3-1为本发明密度梯度离心纯化海绵原细胞示意图 （用 Ficoll和泛影葡胺联合 配制的密度梯度层）。
图 3-2为本发明密度梯度离心后海绵原细胞分布情况示意图。
图 4-1为本发明密度梯度离心后示意图 （C5层的原细胞（χ400) )。
图 4-2为本发明分离纯化后 BrdU免疫组织化学染色图 （原细胞中 BrdU .阳性细 胞（χ400))。
图 4-3为本发明分离纯化后 PCNA免疫组织化学染色图 （原细胞 PCNA阳性细 胞（χ400))。
图 5 为本发明混合海绵细胞处理各步骤所得海绵原细胞的百分比及其 BrdU和
PCNA百分比 （MC为混合细胞； DC为差速离心； DA为差速聚集； DAD为差速黏 附； DGC为密度梯度离心）。
图 6为本发明不同孵育时间对原细胞摄取 BrdU的影响图示。
图 7为本发明混合细胞培养与原细胞纯化培养的体外生长动力学曲线（其中：细 胞生长情况用 MTT活性值表示， MTT值代表细胞数量和细胞活力， 相对细胞生长 指数 =第 N天的 MTT值 /第 1天的 MTT值）。
图 8为本发明海绵混合细胞与纯化原细胞体外培养的生长动力学曲线（细胞生长 情况用细胞数量表示）。
图 9-1为本发明采用南海海绵原细胞分离过程各步骤所得细胞形态图（制备的混 合细胞）。 图 9-2为本发明采用南海海绵原细胞分离过程各步骤所得细胞形态图（低速离心， 经 500rpm， lOmin低速离心后所得海绵细胞）。
图 9-3为本发明采用南海海绵原细胞分离过程各步骤所得细胞形态图（差速聚集 后， 形成的细胞聚集体）。
图 9-4为本发明采用南海海绵原细胞分离过程各步骤所得细胞形态图（原细胞从 黏附于培养瓶底部的上皮细胞层上解离下来）。
图 9-5为本发明采用南海海绵原细胞分离过程各步骤所得细胞形态图（经密度梯 度离心后分离纯化的一种未知类型的皮质细胞）。
图 9-6为本发明采用南海海绵原细胞分离过程各步骤所得细胞形态图（经密度梯 度离心后分离纯化的原细胞）。 . .
图 10-1为分离南海海绵原细胞时经 10%蔗糖速度沉降后的细胞分布情况。
图 10-2为分离南海海绵原细胞时经蔗糖密度梯度离心后的细胞分布情况。
图 10-3为本发明采用南海海绵原细胞分离过程各步骤所得细胞形态图（经 10% 蔗糖沉降后所得底层细胞）。
图 10-4为本发明南海海绵混合细胞与纯化原细胞体外培养生长曲线。
图 11为本发明比较例用 MTT法跟踪混合细胞体外培养过程的细胞生长情况。 图 12-1 为本发明比较例混合细胞体外培养过程中发生的霉菌污染情况照片 （可 见到霉菌菌丝缠绕于海绵细胞团之间 λ
图 12-2为本发明比较例混合细胞体外培养过程中发生的细菌污染情况照片 （其 中细菌和原生动物吸收 ΜΤΤ后变兰紫色， 而细胞仍为棕黄色）。 具体实施方式 ,
实施例 1
本实施例取中国黄勃海繁茂膜海绵， 经预处理后通过使用四步耦联分离， 对海绵 多能干细胞（即原细胞）进行有效的分离和高度纯化， 然后对原细胞进行培养与并考 察其代谢活性物质的情况； 具体如下：
1. 预处理 （海绵细胞离散一混合海绵细胞的制备）
将 2~3g新鲜釆集健康繁茂膜海绵组织块， 用无菌且含庆大霉素 （400ug/ml) 的 天然海水 （CMFSW, 含 460 mM NaCl， 7mM Na2S04, lO mM KCl, lOmM Hepes, pH 8.0)洗涤 3遍， 以除去表面微生物及其他共生物后， 切成 l~2mm3小块， 4°C保存 8小时， 用含 25ppm CuS04的 CMFSW浸泡 3小时， 再用 CMFSW洗涤 3次， 每次 2 小时， 以 160rpm的速度振荡离散出海绵细胞， 300目尼龙滤网过滤， 离心 1000rpm， lOmin, 用含 2mM EDTA (30ml的 EDTA中， 加入 3mg/ml溶菌酶 lml) 的 CMFSW 将细胞重悬， 获得混合海绵细胞（见图 1-1 )， 用血细胞计数板计数混合细胞中原细胞 的百分含量， 留取样品考察海绵细胞中增殖细胞核抗原（PCNA)阳性率（见图 1-3 )， 再将部分样品与 BrdU共孵育， 考察混合细胞中 BrdU阳性细胞百分比 （见图 1-2)， 余留细胞用于下一步分离。
2. 原细胞的分离纯化
2.1差速离心法一富集包括原细胞在内的大体积海绵细胞
将取步骤 1余留的混合海绵细胞用 CMFSW将细胞密度调整为 5xl07/ml，再以较 小转速离心， 300rpm， 15min, 弃去上清液， 保留细胞沉淀， 得到包括原细胞在内的 较大体积的海绵细胞（见图 1-4); 计数其中原细胞百分比， 留取部分细胞考察 PCNA (见图 1-6) 与 BrdU (见图 1-5 ) 的阳性细胞百分比， 其余细胞用于下一步分离。
2.2差速聚集法一富离聚集能力强的海绵细胞 （以胶原细胞和原细胞为主的混合 体）
将步骤 2.1所得细胞沉淀， 用 CMFSW洗涤 2次以除去残存的 EDTA, 调整细胞 密度为 10xl07/ml， 于 100ml容积的玻璃培养瓶中培养， 实际培养体积为 10ml/瓶， 18°C， 8小时， 振荡培养， 20转 /min， 使原细胞充分参与聚集， 形成细胞聚集体 （见 图 2-1 )，弃去未参与聚集的游离海绵细胞， 留取部分细胞样品计数细胞聚集体中的原 细胞百分含量 （见图 2-7) 并检测 PCNA (见图 2-9) 和 BrdU (见图 2-8 ) 阳性率， 其余细胞用于下一步分离。
2.3 差速黏附法初步分离原细胞一初步将胶原细胞和原细胞分离
改用 ASW (artificial sea water,人工海水，含 460 mM NaCl, 7mM Na2S04, 10 mM KC1, 10 mM CaCl2, 50 mM MgCl2, lOmM Hepes, ρΗ δ.Ο) 继续培养细胞聚集体， 8 小时，使之贴壁，待聚集体中的胶原细胞和上皮细胞充分贴壁分布于细胞聚集体底部， 并进行活跃迁移、 铺展达 80%汇合时 （见图 2-5)， 停止培养， 用 CMFSW洗去 ASW 后， 用含 10mM EDTA的 CMFSW将分布于细胞聚集体上层的原细胞离散下来 （见 图 2-6)， 镜下控制 EDTA作用的时间， 22°C下， 处理时间为 5min， 以防止作用时间 过长使过多的胶原细胞脱壁；在使用 EDTA处理海绵细胞时，为减少对海绵细胞的化 学损伤， 本实施例使用 2mM EDTA处理 lOmin的效果最合适。 将离散下来的原细胞 聚集体充分离散， 吹打均勾 （见图 2~10)， 得初步分离的原细胞一初步将上皮细胞 / 胶原细胞和原细胞分离； 取样计数其中的原细胞百分比并考察 PCNA (见图 2-12)和 BrdU (见图 2-11 ) 阳性率， 其余细胞用于下一步分离。
2.4密度梯度离心纯化原细胞一进一步将原细胞与其他细胞分开
1 ) 将步骤 2.3所得初步离散的原细胞密度调节为 10xl07/ml， 备用。
2) 配制梯度溶液
用 CMFSW将 Ficoll粉末配制成 3%Ficoll (p=1.0276g/ml, 16°C )， 并将 76%的泛 影葡胺原液稀释成为 38% (p=1.1486g/ml， 16°C ), 经过计算， 配制密度为 1.04， 1.05， 1.06， 1.07， 1.08, 1.09， 1.10g/ml的梯度液 (见图 3-1 );
3 ) 将调节密度后的初步离散的原细胞离心 用 15ml试管时， 每层梯度液为 lml， 上样量为 lml， 总细胞数量为 2> 107； 用水 平离心机， lOOOrpm, 20min, 进一步将原细胞与其他细胞分并。
待离心完毕， 细胞分层后， 用滴管将各层细胞取出 用 CMFSW洗涤 2次， 去除 密度梯度介质， 镜下观察各层细胞形态， 原细胞分布于密度为 1.07和 1.08两密度层 之间 （见图 3-2)， 取样计数原细胞百分比 （见图 4-1 ) 并考察 PCNA (见图 4-3 ) 和 BrdU (见图 4-2) 阳性率， 部分细胞用于细胞培养观察体外生长情况。
3. 海绵原细胞增殖活力的检测
1 ) BrdU标记及其免疫组织化学染色
从上述各步骤所得海绵细胞中取样， 计数其中的原细胞含量及细胞密度， 按照 106个细胞对应加入 0.5ml BrdU ( 1 : 1000) 进行孵育， 18°C， 24小时， 考察各步骤 所得海绵细胞的 BrdU阳性率。 具体的 BrdU阳性率的检测方法， 按照 Roche公司的 BrdU标记和检测试剂盒说明书进行。
同时考察了细胞与 BrdU共孵育时间对细胞摄取 BrdU的影响， 见图 6。
2) PCNA的表达
应用增殖细胞核抗原 （PCAN) 这一指标来考察海绵原细胞增殖能力， 实验数据 表明， 随着分离过程的进行， 所分离细胞中原细胞的比例逐渐增加， PCNA阳性细胞 的比例也随之增加 （见图 5所示）， 说明所分离的细胞具有较强的增殖能力， 从而证 明所分离的细胞是海绵原细胞。
4. 海绵细胞的体外增殖培养与代谢考察
以黄渤海繁茂膜海绵为例； 按 8xl06cellS/ml接种混和细胞， 培养体积 2ml, 每隔
2天更换 1/2量的改进海水培养基液， 在 18°C恒温光照培养箱中静置或振荡培养， 光 源为日光灯。
改进人工海水培养基液为： 在海水基础培养基中添加海绵细胞间质液提取物。 其中： 海水基础培养基为： 460 mM NaCl (氯化钠）， 7mM Na2S04 (硫酸钠）， lO mM KCl (氯化钾）， 10 mM CaCl2 (氯化钙）， 50 mM MgCl2 (氯化镁）， 20mM Tris (三羟基氨基甲垸）， 30mM Na2SiO3.9H2O (硅酸钠）， 60μΜ FeC6H507.5H20 (柠檬 酸铁）， 4μΜ ZnCl2 (氯化锌）， 5μΜ Vit C (维生素 C)， 10μΜ L-Glutamine (谷氨酰 胺）， 10μΜ L-asparagin (天冬酰胺）， ΙΟμΜ glycine (甘氨酸）， 20μΜ glycose (葡萄 糖）， ImM sodium pyruvate (丙酮酸钠）， pH 8.0。
所述海绵细胞间质液提取物制备： 取 10g新釆集海绵块， 浸泡于 100g CMFSW 中， 由于缺钙海绵组织出现"骨质疏松"状态，胞间间质分子就会释放到 CMFSW中， 平均 2小时更换 90%的 CMFSW, 共 5次， 共收集 1L浸出液， 0.22μπι滤膜无菌过 滤， 获得用于海绵细胞培养浸出液； 浸出液与海水细胞基础培养基等比例混合使用。
按步骤 1制备海绵原细胞， 并应用上述改进海水培养基进行体外培养， 考察细胞 增殖和代谢动力学变化， 同时与海绵混和细胞培养进行比较。海绵原细胞和混合海绵 细胞的体外培养增殖情况见图 7和图 8。 培养中， 检测细胞数量禾代谢产物表达。 细 胞培养过程中， β-胡萝卜素的合成可达到 0.01 mg/ ( 106 cells/ml )(, ' 5a-c olesterol-3 β-ol 可达到 0.1mg/5xl06 cells/ml。可见， 釆用本发明的同时还可以通过海绵原细胞体外大 规模培养来生产活性物质。
实施例 2
南海海绵 （种属未鉴定） 的原细胞纯化
与黄海繁茂膜海绵原细胞的分离原理和方法基本相同，但由于不同种属海绵细胞 间在沉降系数、聚集贴附能力上不同， 所以将上述方法稍做改进， 可以得到该种南海 海绵的干细胞。 不同之处在于：
步骤 1 :采用水族槽暂养 14天的南海健康海绵组织块，用含庆大霉素（800ug/ml) 的天然海水洗涤除去表面微生物及其他共生物后， 切成 2~3mm3小块， 再用 CMFSW ( 986mM NaCl, 其他成分同上）洗涤 4次， 每次 3小时， 加入含 EDTA的 CMFSW 振荡离散后过滤， 离心 2000rpm,10min， 用含 2mM EDTA的 CMFSW将细胞重悬， 获得混合海绵细胞。 · . 步骤 2.1 : 取步骤 1混合海绵细胞用 CMFSW将细胞密度调整为 2x l07/ml， 制备 混合细胞 （见图 9-1 )， 将混合海绵细胞经 500rpm,10min后， 富集得到体积较大的皮 质层和髓质层海绵细胞 （见图 9-2)。
步骤 2.2:将步骤 2.1所得细胞沉淀，用 CMFSW洗涤后调整细胞密度为 5>< 107/ml， 于玻璃培养瓶中用培养， 25 V， 12 小时， 振荡， 弃去未参与聚集的游离海绵细胞， 细胞聚集体用于下一步分离 （见图 9-3)。
步骤 2.3 : 改用 ASW (986mM NaCl, 其他成分同上） 继续培养细胞聚集体， 10 小时，使之贴壁，待聚集体中的胶原细胞和上皮细胞充分贴壁分布于细胞聚集体底部， 并进行活跃迁移、 铺展达 70 %汇合时， 停止培养， 用 CMFSW洗去 ASW后， 用含 5mM EDTA的 CMFSW将分布于细胞聚集体上层的原细胞离散下来（见图 9-4)， 镜 下控制 EDTA作用的时间， . 18°C下， 处理时间为 l Omin (如使用 lOmM EDTA, 则处 理 5miri)。 将离散下来的原细胞聚集体充分离散， 吹打均匀， 得初步分离的原细胞一 初步将原细胞和其他细胞分离；
步骤 2.4:
1 ) 将步骤 2.3所得初步离散的原细胞密度调节为 5x l07/ml;
2 )先将步骤 2.1分离得到的海绵细胞经低浓度蔗糖（本实施例为 10% ) 进行速 度沉降， 将细胞按沉降速度分为三部分 （见图 10-1 )， 其中最上层为体积较小的皮质' 层细胞； 第二层为体积较大的皮质和髓质层细胞以及很少一部分原细胞， 约占 10%; 第三层即沉降速度最块一层为体积较大的非原细胞与原细胞的混合物， 其中原细胞， 约占 65%; 取步骤 2.2沉降速度最快的一部分细胞（见图 10-3 )继续进行蔗糖密度梯 度离心， 蔗糖浓度梯度为 20%, 30% , 40%, 50%, 60% (见图 10-2)。 原细胞则分布 于密度最高的底层， 分离纯化后的原细胞， 纯度 >80%。 利用相 方法可以分离纯化 得到该种海绵的另外一种细胞， 该细胞类型还未作最后鉴定 （见图 9-5)。
3 )采用 50ml试管进行密度梯度离心时， 则每层梯度液为 8ml， 上样量为 5ml， 总细胞数量为 l x lO8;收集后的原细胞用 CMFSW洗去密度梯度介质，用水平离心机， 1500rpm, lOmin, 共洗涤 3次， 进一步将原细胞即多能干细胞与其他细胞分开。
步骤 3: 采用步骤 1分离的干细胞进行培养; 细胞接种密度 2x l06 CeUs/ml， 培养 过程中每隔 1天更换 70%的改进海水培养基液，控制培养温度在 25°C (暗培养）; 40rpm 转速下振荡培养； 计数细胞数量， 与海绵混和细胞培养进行比较细胞增殖情况； 所述改进海水培养基液为在海水基础培养基的基础上添加所述生长因子为： 60 g/LNa2Se03， lmg/L二硫苏糖醇（DTT)及 240 g/L谷胱甘肽 （GSH)。
其比较结果参见图 10-4混合细胞与纯化原细胞体外培养生长曲线。
实施例 3
南海海绵 （种属未鉴定） 的原细胞纯化
与实施例 2不同之处在于：
步骤 1： 采用水族槽暂养 7天的南海健康海绵组织块，用含庆大霉素（200ug/ml) 的天然海水洗涤除去表面微生物及其他共生物后， 切成 l~3mm3小块， 再用 CMFSW (986mM NaCl,其他成分同上）洗涤 5次，每次 2.5小时， 加入含 EDTA '的 CMFSW 振荡离散后过滤， 离心 2500rpm， 20min, 用含 3 mM EDTA的 CMFSW将细胞重悬， 获得混合海绵细胞。
'步骤 2.1： 取步骤 1混合海绵细胞用 CMFSW将细胞调整密度后制备混合细胞， 将混合海绵细胞经 600rpm,20min后，富集得到体积较大的皮质层和髓质层海绵细胞。
步骤 2.2:将步骤 2.1所得细胞沉淀，用 CMFSW洗涤后调整细胞密度为 7x l07/ml， 于玻璃培养瓶中用培养， 20°C， 10小时， 振荡， 弃去未参与聚集的游离海绵细胞， 细胞聚集体用于下一步分离。
步骤 2.3 : 改用 ASW (986mM NaCl， 其他成分同上） 继续培养细胞聚集体， 10 小时，使之贴壁，待聚集体中的胶原细胞和上皮细胞充分贴壁分布于细胞聚集体底部， 并进行活跃迁移、 铺展达 75 %汇合时， 停止培养， 用 CMFSW洗去 ASW后， 用含 6mM EDTA的 CMFSW将分布于细胞聚集体上层的原细胞离散下来，镜下控制 EDTA 作用的时间， 20°C下， 处理时间为 5min; 如使用 5mM EDTA， 则处理 7min。 将离散 下来的原细胞聚集体充分离散，吹打均匀，得初步分离的原细胞一初步将胶原细胞和 原细胞分离；
步骤 2.4:
1 )将步骤 2.3所得初步离散的原细胞密度调节为 5x l07/ml;
2) 先将步骤 2.1分离得到的海绵细胞经低浓度蔗糖 （本实施例为 20% )进行速 度沉降， 再进行蔗糖密度梯度离心， 蔗糖梯度为 12% , 24% , 36% , 48% , 60%。 3 )采用 50ml试管时，则每层梯度液为 10ml，上样量为 3ml，总细胞数量为 2> 108； 收集后的原细胞用 CMFSW洗去密度梯度介质， 用水平离心机， 500rpm， 15min, 共 洗涤 4次， 进一步将原细胞与其他细胞分开。
步骤 3 : 控制培养温度在 25°C (暗培养， 南海海绵一般都得暗培养， 因为其生长 的海水较深）；
改进海绵细胞基础培养基成份可以在海水基础培养基的基础上添加所述分化控 制因子为： 20 μ M/ml羟基脲及 10 μ M/ml视黄酸。
采用步骤 1分离的原细胞进行培养；细胞接种密度 3x l07 Cells/ml，培养过程中每 隔 1天更换 70%的改进海水培养基液， 控制培养温度在 25°C， 20rpm转速下振荡培 养； 计数细胞数量， 与海绵混和细胞培养进行比较细胞增殖情况。
本发明基于海绵原细胞是海绵组织中的多能干细胞， 具有较强的增殖能力的理 论， 首次提出海绵干细胞的体外培养的技术路线， 通过发明的四步耦合分离方法， 实 现了海绵中原细胞的有效分离和高度纯化。 在获得高度纯化的海绵干细胞的基础上， 进一步建立了海绵干细胞体外培养的工艺条件， 包括培养的理化条件、培养基、调控 分化和生长的关键因子、培养方式和培养的生物反应器等核心技术，实现了海绵细胞 的体外培养和稳定增殖，并在所试验的海绵体系中能有效生产海绵生物活性物质。本 发明为解决重要药源生物、海绵生物活性物质的生产提供了全新的技术途径，对海洋 药物，尤其是海绵药物的产业化具有重大意义， 同时也为海洋无脊椎动物的细胞生物 学等基础研究提供了重要手段，具有重要的理论意义和应用价值。预期的商业前景将 极为广阔。
本发明改进海绵细胞基础培养基成份还可以在人工海水培养基的基础上添加生 长因子为： 可溶性胆固醇，氢化可的松，胰岛素， 转铁蛋白，植物血球凝集素（PHA) 之一或其组合； 或分化控制因子视黄酸、 羟基脲之一。
比照例
常规技术培养混合海绵细胞的生物量变化 '
采用常规化学离散法获得的混合细胞， 并使其直接在 ASW或天然海水中成团， 为防止微生物污染加入 100 U/ml庆大霉素或青霉素 100U/ml， 链霉素 lOO g/ml, 两 性霉素 3 g/ml进行培养， 用 MTT法检测细胞的生长情况， 结果参见图 11所示， 细 胞培养体系从第 4天开始 MTT检测值开始增高， 到第七天达到初始 MTT值的 8倍 以上， 经镜下检査， 发现了大量的细菌和霉菌（见图 12-1、 12-2)生长， 可以断定所 测得的生物量的异常增长为细菌和霉菌的生长。第四天的极大生物量经镜下检査为大 量的原生动物生长。因此可以说明，采用常规海绵细胞离散成团技术培养的混合细胞 团中的污染问题严重干扰海绵细胞的培养和生长， 使海绵细胞团难以正常生长和增 殖。

Claims

权 利 要 求 书
1. 一种海洋海绵多能干细胞的纯化与培养方法， 其特征在于： 将经过蓣处理的 海绵细胞依次进行差速离心、差速聚集、差速黏附和密度梯度离心， 根据海绵细胞的 大小和粘附特性进行分离、 纯化；
1 )预处理 （制备混合海绵细胞）
取新鲜或经水族槽暂养 7~14天的健康海绵组织块，用含 200~800μ§ΛΏ1庆大霉素 的天然海水洗涤， 剔除杂质； 再将组织块切割成块， 浸泡于无钙镁海水中洗涤， 振荡 离散出海绵细胞， 过滤， 离心； 再用含 l~2mM乙二胺四乙酸的无 #5镁海水制备细胞 悬液，获得混合海绵细胞，待下一步分离；其中离心速度为： 1000~3000rpm， 10~20min;
2)海绵多能千细胞的纯化 - 2.1差速离心
.将取步骤 1获得的混合海绵细胞用无钙镁海水调整细胞密度，再以 300~600 rpm， 10~20min转速离心， 弃上清液， 保留细胞沉淀， 得到富集包括原细胞在内的大体积 海绵细胞， 待下一步分离；
2.2差速聚集
将步骤 2.1所得细胞沉淀， 用无钙镁海水洗涤以除去残存的乙二胺四乙酸， 调整 细胞密度， 于容器中用无钙镁海水在 18~25°C下培养 8〜12小时， 使所述原细胞充分 参与聚集， 形成细胞聚集体， 然后弃去未参与聚集的游离海绵细胞，其余细胞用于下 一步分离；
2.3 差速黏附
用人工海水继续培养所述细胞聚集体使之贴壁， 待细胞聚集体中的胶原细胞和上皮 细胞完全贴壁分布于细胞聚集体底部， 并进行活跃迁移、 铺展达 70~80%汇合时， 停止 培养， 再用无钙镁海水洗去人工海水后， 用含 2~10mM 乙二胺四乙酸的无钙镁海水将 分布于细胞聚集体上层的原细胞离散下来， 在 18~22°C下， 镜下控制乙二胺四乙酸作用 的时间 5~10ηώι;得初步分离原细胞， 即初步将胶原细胞和原细胞分离，待下一步分离；
2.4密度梯度离心
2.4.1将步骤 2.3所得初步分离的原细胞， 调节其密度为 5〜10xl07/ml， 备用；
2.4.2配制梯度液；
2.4.3将调节密度后的初步离散的原细胞进行密度梯度离心
用试管， 每层梯度液为 l~10ml， 上样量为 l~5ml， 总细胞数量为 2~20><107 ; 收 集后的原细胞用无钙镁海水洗去密度梯度介质， 用水平离心机， 500~1500rpm， 5~20min, 共洗涤 2〜4次， 从而进一步将原细胞即多能干细胞与其他细胞分开。
2. 按权利要求 1所述海洋海绵多能干细胞的纯化与培养方法， 其特征在于： 所
]2 述步骤 2.3中最好用含 2~5mM 乙二胺四乙酸的无钙镁海水将分布于细胞聚集体上层 的原细胞离散下来。
3. 按权利要求 1所述海洋海绵多能干细胞的纯化与培养方法， 其特征在于： 步 骤 2.4 中所述梯度液的配制是： 用无钙镁海水将 Ficoll 粉末配制成 3~9%Ficoll ( p=l.0276-1.0445g/ml, 16~20°C ) , 并将 76%的泛影葡胺原液稀释成为 34~38% (p=1.1379~1.1486g/ml， 16~20°C )， 配制密度为 1.04， 1.05, 1.06， 1.07， 1.08， 1.09, 1.10g/ml的梯度液。
4. 按权利要求 1所述海洋海绵多能干细胞的纯化与培养方法， 其特征在于： 步 骤 2.4中所述梯度液的配制还可以是： 用蔗糖密度梯度离心， 蔗糖浓度梯度为 12~24 % , 24-36% , 36-48% , 48-52% , 58~60%。
5. 按权利要求 1所述海洋海绵多能干细胞的纯化与培养方法， 其特征在于： 步 骤 2.4 .中所述将调节密度后的初步离散的原细胞进行密度梯度离心，用 15mH式管时, 每层梯度液为 l~2ml，上样量为 l~2ml，总细胞数量为 2~5χ107;或采用 50ml试管时， 每层梯度液为 5~10ml， 上样量为 3~5ml， 总细胞数量为 1~2><108。
6. 按权利要求 1所述海洋海绵多能干细胞的纯化与培养方法， 其特征在于： 加 设海绵细胞的体外增殖培养与代谢考察步骤，具体：采用步骤 1分离的多能干细胞进 行培养； 细胞接种密度 2xl06~3xl07 cells/ml， 培养过程中每隔 1~2天更换 50~70%的 改进人工海水培养基液， 控制培养温度在 18~25°C， 在静置或 20~40rpm转速下振荡 培养； 计数总细胞、 原细胞数量， 捡测海绵细胞中增殖细胞核抗原阳性率， 并与海绵 混和细胞培养进行比较细胞增殖情况；
其中：所述改进人工海水培养基液成分为：海水基础培养基添加海绵细胞间质液 提取物、 其它生长因子或分化控制因子。
7. 按权利要求 6所述海洋海绵的干细胞的纯化与培养方法， 其特征在于： 所述 控制培养可根据海绵种类进行光照或暗培养。
8. 按权利要求 6所述海洋海绵多能干细胞的纯化与培养方法， 其特征在于： 所 述海绵细胞间质液提取物制备： 取新釆集健康海绵块， 以 1/10~1/20的重量 /体积比浸 泡于无钙镁海水中， 平均 2-4小时更换 80~100%的无钙镁海水， 共 4~6次， 收集全部 浸出液， 无菌过滤， 获得用于海绵细胞培养的浸出液； 按照浸出液占 25 %~75 %的比 例与海绵细胞基础培养基混合使用。
9. 按权利要求 6所述海洋海绵多能干细胞的纯化与培养方法， 其特征在于： 所 述生长因子为： 5-20mg/ml可溶性胆固醇， 5-2(^g/ml氢化可的松， 5-10μ§/πι1胰岛素， 5-l(^g/ml转铁蛋白， 6-10xl(T5g/L Na2Se03， 15-5(^g/ml植物血球凝集素 （PHA)， 100-2.00μ§/ιη1二硫苏糖醇、 2-8xl(T4g/L谷胱甘肽之一或其组合。
10. 按权利要求 6所述海洋海绵多能千细胞的纯化与培养方法， 其特征在于： 所 述分化控制因子为： 5-50 M/ml羟基脲、 5-20 μ M/ml视黄酸之一或其组合。
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