CN1332024C - 3D culture method for micro carrier of sponge cell - Google Patents

3D culture method for micro carrier of sponge cell Download PDF

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CN1332024C
CN1332024C CNB200410020708XA CN200410020708A CN1332024C CN 1332024 C CN1332024 C CN 1332024C CN B200410020708X A CNB200410020708X A CN B200410020708XA CN 200410020708 A CN200410020708 A CN 200410020708A CN 1332024 C CN1332024 C CN 1332024C
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microcarrier
cell
sponge cell
sponge
culture method
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CN1706937A (en
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张卫
赵权宇
虞星炬
金美芳
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Dalian Institute of Chemical Physics of CAS
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Abstract

The present invention relates to a culture method of an oceanic invertebrate cell, more specifically to a three-dimensional culture method of a microcarrier of a sponge cell, which comprises the steps: 1) a microcarrier and a sponge cell are vaccinated in improved artificial sea water for culture, and Na+, SO<4><2->, HCO3-, K+, Tris HCL, Cl-, Mg2+, Ca2+, SiO<3><2->, Fe3+, Zn2+, C6H5O<7><3->, pH 7.6 to 8.2 and PHA are contained in the water solution of the improved artificial sea water; 2) after the sponge cell is cultured for 5 to 8 days, organic carbon sources, organic nitrogen sources and vitamin C are added to the improved artificial sea water so as to form a new culture solution system; 3) after the step 2) is carried out, the culture solution is exchanged every 2 to 3 days, the organic carbon sources, the nitrogen sources and the vitamin C are added, and the sponge cell can normally grow under the condition. The present invention has the advantages that the large-scale production of medicine resources can be realized, the materials can be widely selected, and the application prospects are good.

Description

A kind of microcarrier three-dimensional culture method of sponge cell
Technical field
The present invention relates to the cultivation of marine invertebrate cell, specifically a kind of microcarrier three-dimensional culture method of sponge cell.
Background technology
Sponge is the marine invertebrate of minimum grade, genus polyporus animal door (Porifera), battalion's filter food, set growth.The secondary metabolite that has been found that sponge has biological activitys such as anti-inflammatory, antitumor, anti-HIV.Adopting modern biotechnology is problem (the document 1.Belarbi EH that receive much attention in the world at present at these active substances of produced in vitro, G ó mez AC, Chisti Y, Camacho FG, Grima EM.Producing drugs from marine sponges, Biotechnology Advances, 21 (7), 585-598,2003).The bottleneck problem that the research of sponge natural product developing drugs runs into is the undersupply in medicine source.Solution route comprises sea farming, organic synthesis, cell cultures etc.(document 2.Custodio MR such as Custodio, Prokic I, Steffen R, Koziol C, Borojevic, Br ü mmer F, NickelM, M ü ller WEG.Primmorphs generated from dissociated cells of the spongeSuberites domuncula:a model system for studies of cell proliferation and celldeath, Mechanisms of Ageing and Development, 105 (1-2), studies show that 45-59,1998) forms aggregate---cell mass by cell-cell adhesion, can recover the telomerase activation of sponge cell, make cell have multiplication potentiality.Some scholars form cell mass to different sponges and have done further research, are considered to one of the most promising sponge cell isolated culture method at present.(document 3.Wei Zhang such as Zhang Wei, Xiaoying Zhang, Xupeng Cao, Junyi Xu, QuanyuZhao, Xingju Yu, Meifang Jin, Maicun Deng.Optimizing the formation of Invitro sponge primmorph from Chinese sponge Stylotella agminata (Ridley) .J.Biotechnol.100:161-168,2003) cell mass that South China Sea is sticked up prominent pin (Stylotell agminataRidley) sponge is cultivated and has also been done more deep research.
Microcarrier is widely used in the extensive isolated culture of zooblast, and the microcarrier of having developed comprises solid and porous microcarrier two classes, can adapt to the attaching and the high-density culture of dissimilar cells.The material of microcarrier is more, modified glucan, glass, collagen etc.Wherein, Cytodex series microcarrier and glass microcarrier are used wider.Hillex is the novel microcarrier of Solohill company exploitation, goes for the growth of zooblast in serum-free and protein-free medium.These microcarriers all are Land mammals and the isocellular high-density culture exploitation of insect, whether the marine invertebrate cell are suitable for be still waiting research.Mcmahon (document 4.Mcmahon P.System for the cell cultureand cryopreservation of marine invertebrates.US 6054317,2000) polystyrene series microcarrier is used for the isolated culture of sponge cell, has also used the serum of gastropod.Discrete cell easily comes off; The artificial seawater prescription, cell mainly exists with the cell mass form; Gastropod serum obtains difficulty, costs an arm and a leg, and is not suitable for scale operation, but this work provides new thinking for the isolated culture of sponge cell.The attaching of cell on the microcarrier surface is the committed step of using microcarrier.Zhao (document 5.Zhao QY, Jin MF, M ü iller WEG, Zhang W, Yu XJ, Deng MC.Attachment of Marine Sponge Cells of Hymeniacidon perleve on Microcarriers, Biotechnology progress, 19 (5), 1569-1573,2003) etc. investigated the discrete cell of Yellow Sea of China film sponge in great numbers (Hymeniacidon perleve) at Cytodex 3, the influence of attaching character on three kinds of microcarriers of glass microcarrier and Hillex and different vaccination condition to attaching.The emphasis of this research is by the effect of discrete cell and microcarrier, selects suitable microcarrier material.The result shows that the attaching of Yellow Sea of China film sponge in great numbers cell on glass microcarrier surface obviously is better than Cytodex 3 and Hillex.A key during the sponge cell long-period is cultivated is a substratum, comprises physical condition and nutritional conditions such as osmotic pressure.Substratum is one of research emphasis of sponge cell isolated culture for the growth and the metabolism of cell provide nutrient and suitable envrionment conditions.At present the substratum of exploitation mainly is Mammals and insect cell line, up to now, the research of relevant sponge cell culture medium also seldom, developing targetedly at the different genera sponge, substratum is very important.Willoughby (document 6.Willoughby R, Pomponi SA.Quantitative Assessment of Marine Sponge Cells in Vitro:Development Of Improved Growth Medium.In Vitro Cell.Dev.Bio1.-Animal36:194-200,2000) studied Teichaxinella morchella sponge (Porifera, Demospongiae, Axinellida, the Axinellidae) influence of glutamine, selenium and calf serum in the substratum.(document 7.Zhang XY such as Zhang XY, Pennec GL, Steffen R, M ü ller WEG, Zhang W.Application of a MTT Assay for Screening Nutritional Factors inGrowth Media of Primary Sponge Cell Culture.Biotechnology progress, 20 (1), 151-155,2004) investigated several trophic factor (SiO 3 2-, Fe 3+, glutamine, pyruvic acid etc.) to the influence of Hermit crab suberite Suberites domuncula growth.Muller WEG proposes the cell mass culture technique (document 8.Muller WEG Production of primmorphs fromdissociated cells of sponges and coral.US 6664106 B1) of sponge cell; But it does not provide the optimization culture medium prescription.In addition, the sponge of different genera has different nutritional needs, and it is necessary developing general and special culture media.
Summary of the invention
The sponge cell microcarrier three-dimensional culture method that the object of the present invention is to provide a kind of sponge cell mass to attach and cultivate on microcarrier surface is for the isolated culture of sponge cell provides a kind of feasible new technology; Under certain conditions, with cell mass firm be attached to the microcarrier surface, more help keeping the telomerase activation and the proliferative of sponge cell.
For achieving the above object, the technical solution used in the present invention is:
Choosing of microcarrier: microcarrier can be selected solid microcarriers such as glass microcarrier, Cytodex 3, also can select the solid or porous microcarrier of Composite Preparation such as ImmobaSil series, Cytoline series porous microcarrier, gelatin, gelatin+polylysine or gelatin+chitosan.The inoculation condition of solid microcarrier is 5～20g/L (nutrient solution), and the inoculation condition of porous microcarrier is 2～10g/L (nutrient solution).
The cultivation of sponge in improving artificial seawater: seawater is formed and is comprised: Na +300～500mM, SO 4 2-7.0mM, HCO 3 -0.2mM, K +10.0mM, Tris HCl 20.0mM, Cl -300～500mM, Mg 2+50mM, Ca 2+10.0mM, SiO 3 2-5～150 μ M, Fe 3+30～120 μ M, Zn 2+0.5～4 μ M, C 6H 5O 7 3-30～120 μ M, pH 7.6～8.2.The interpolation concentration of PHA is 1.5～2.5%.
After cultivating 5～8 days, add carbon source (glucose, sorbyl alcohol or Sodium.alpha.-ketopropionate), nitrogenous source (comprising glycine, l-asparagine and glutamine) and ascorbic concentration 5～20 μ M, changed 50～80% of nutrient solution volume afterwards every 2～3 days, replace and add carbon source and nitrogenous source makes its ultimate density reach 20～50 μ M with improved artificial seawater, the sponge cell can carry out normal growth with this understanding.
The added ingredients of substratum also may comprise 0.01～10.0% DMEM or the canavaline (ConA) of RPMI 1640,5～15 μ g/L.
The sponge cell inoculation scope that is fit to is 1 * 10 6～9 * 10 7Cells/ml.
The present invention has following advantage:
1. the medicine source can be mass-produced.The present invention proposes the microcarrier of cell mass and cultivates, and cell aggregation is adherent firmly, when guaranteeing the sponge telomerase activation, for sponge cell proliferation provides wall-attached surface, helps the large scale culturing of sponge cell.
2. optional material is more extensive.Behind the medium optimization of the present invention, the cell attachment ability strengthens, and there is the microcarrier the selected range extension of sponge cell cultures in cell after forming aggregate with the form that is attached at the microcarrier surface; The microcarrier that can be used for the sponge cell cultures also comprises porous microcarrier (commercial microcarrier: solid microcarrier such as glass, and ImmobaSil series and Cytoline series porous microcarrier) except that solid microcarrier; Material expands organosilicon material, gelatin and modified composite material thereof to.
3. the present invention proposes the microcarrier culture technique of sponge cell mass; Be that aggregate form with the sponge cell carries out microcarrier and cultivates; The optimization substratum that suitable microcarrier is cultivated has been proposed; Comprise elements such as zinc in the substratum, and somatomedin and inhibition disintegrating agent; And adopt the method for progressively the adding control of nutritive ingredient to pollute.
4. application prospect is good.The invention belongs to the marine invertebrate field of cell culture, can realize that the large scale culturing of sponge cell is produced the sponge secondary metabolite, for the exploitation of marine drug provides medicine source production technology.
Description of drawings
Fig. 1 is the light microscopic photo (* 100 of the attaching process of film sponge cell in great numbers on Glass Beads; Inoculation A:0h; B:2h; C:12h; D:25h);
Fig. 2 is the light microscopic photo (A: * 40 of the cultivation of film sponge cell in great numbers in improving artificial seawater; B: * 400);
Fig. 3 is the light microscopic photo (* 250) that the artificial seawater of film sponge in great numbers is cultivated;
Fig. 4 is that the ImmobaSil FS of film sponge cell in great numbers cultivates the SEM photo;
Fig. 5 is that the ImmobaSil FS of film sponge cell in great numbers cultivates light microscopic photo (* 400).
Embodiment
Embodiment 1
1) film sponge 1 * 10 in great numbers 7Cells/ml, volume of culture 4ml, 18～22 ℃, the cultivation of sponge cell in improved artificial seawater: seawater is formed and is comprised: Na 2SO 40.9943g/L, NaHCO 30.0168g/L, Tris-HCl 2.4218g/L, KCl 0.7455g/L, Na 2SiO 39H 2O 0.0085g/L, FeC 6H 5O 75H 2O 0.0201g/L, NaCl 20.0g/L, ZnCl 20.00054g/L, MgCl 26H 2O10.165g/L, CaCl 21.1099g/L.The interpolation concentration of PHA is 2.5%.Choose the glass microcarrier, inoculation condition 15g/L (improvement artificial seawater).
2) after cultivating 7 days, add glucose 15 μ M, glycine 10 μ M, l-asparagine 10 μ M and glutamine 10 μ M, vitamins C 10 μ M, changed 50～80% of nutrient solution volume afterwards every 2～3 days, replace and add carbon source and nitrogenous source makes its ultimate density reach 50 μ M with improved artificial seawater, ascorbic concentration is at 20 μ M, and the sponge cell can carry out normal growth with this understanding.Add the 80U gentamicin in the nutrient solution simultaneously, the generation that controlling microbial is polluted.
Referring to shown in Figure 1, observe the glass microcarrier culturing process of film sponge cell in great numbers, free cell is attached on the microcarrier very soon; The part free cell comes off, and the part cell forms cell aggregation in nutrient solution; Cell aggregation cognition is attached on the microcarrier, increases with space-time ball rate.This forming process with cell mass is slightly different.Free cell attaches best on the glass microcarrier, and the bigger aggregate that is formed by a plurality of cell aggregations and microcarrier also is occur in the glass microcarrier the fastest.Sponge cell group is more firm is attached to the microcarrier surface, even forms and adhere to and the expansion of cell.
Embodiment 2
Film sponge 1 * 10 in great numbers 7Cells/ml, volume of culture 4ml, 18～22 ℃, the sponge cell is at improved artificial seawater (Na 2SO 40.9943g/L, NaHCO 30.0168g/L, Tris-HCl 2.4218g/L, KCl 0.7455g/L, Na 2SiO 39H 2O 0.0085g/L, FeC 6H 5O 75H 2O 0.0201g/L, NaCl20.0g/L, ZnCl 20.00054g/L, MgCl 26H 2O 10.165g/L, CaCl 21.1099g/L) and 2.5%PHA in cultivate, the sponge cell mass is attached to cultivates the orifice plate bottom, at control medium (Na 2SO 40.9943g/L, NaHCO 30.0168g/L, Tris-HCl 2.4218g/L, KCl 0.7455g/L, NaCl31.5591g/L, MgCl 26H 2O 10.165g/L, CaCl 21.1099g/L) in, cell exists but not adherent (shown in Fig. 2,3) with the form of aggregate.
Embodiment 3
Film sponge cell 3.0～8.0 * 10 in great numbers 6Cells/ml, and inoculate with 3g/L ImmobaSil FS porous microcarrier.In porous microcarrier, discrete cell and cell aggregation can be attached to microcarrier (shown in Fig. 2,3).The artificial seawater of cell consists of Na 2SO 40.9943g/L, NaHCO 30.0168g/L, Tris-HCl 2.4218g/L, KCl 0.7455g/L, NaCl 31.5591g/L, MgCl 26H 2O10.165g/L, CaCl 21.1099g/L.
Embodiment 4
In 96 orifice plates (polystyrene material), add 50 μ l film sponge in great numbers extracting solution respectively, kidney refers to sponge extracting solution, 1% chitosan solution, 0.2% gelatin solution, 0.9% agar, behind 4 ℃ of preservation 48h, take out preparation liquid, CMFSW cleans sponge and extracts liquid film and gelatin film; Add 1%NaOH effect 10h in chitosan film, CMFSW cleans for several times.4 ℃ of preservations of 96 orifice plate refrigerators.Before the use, uv irradiating.Through discontinuous Ficoll density gradient enrichment spongy archaecyte.In the immobilized environment, the migration of cell forms cell aggregation.Different organic substrates can influence the motion of cell, and then influences cell-cell adhesion.Film sponge cell in great numbers can take place to attach and expand on film sponge extract in great numbers, gelatin.Agar can promote sponge cell aggregation, but does not promote to attach.
Embodiment 5
Film sponge about 1 * 10 in great numbers 7Cells/ml, 18～22 ℃ of cultivations, the sponge cell is at improved substratum (Na 2SO 40.9943g/L, NaHCO 30.0168g/L, Tris-HCl 2.4218g/L, KCl 0.7455g/L, Na 2SiO 39H 2O 0.0041g/L, FeC 6H 5O 75H 2O 0.0201g/L, NaCl 23.0g/L, ZnCl 20.000032g/L, MgCl 26H 2O 10.165g/L, CaCl 21.1099g/L PHA 1.5%, chooses Cytodex 3 microcarriers, inoculation condition 5g/L (improvement artificial seawater).
After cultivating 5 days, add 0.1% (v/v) DMEM, glucose 0.0040g/L, glutamine 0.0029g/L, l-asparagine 0.0027g/L, glycine 0.0015g/L and vitamins C 0.0035g/L) the middle cultivation, biomass (mtt assay detection) comparison is according to substratum (Na 2SO 40.9943g/L, NaHCO 30.0168g/L, Tris-HCl 2.4218g/L, KCl 0.7455g/L, NaCl 31.559lg/L, MgCl 26H 2O 10.165g/L, CaCl 21.1099g/L) high by 40%; Changed 50% of nutrient solution volume afterwards every 2～3 days, replace and interpolation carbon source and nitrogenous source make its ultimate density reach 40 μ M with improved artificial seawater, ascorbic concentration is at 10 μ M, and the sponge cell can carry out normal growth with this understanding.Add the 100U gentamicin in the nutrient solution simultaneously, the generation that controlling microbial is polluted.Nutritive ingredient adds step by step and brings in constant renewal in nutrient solution, the effectively controlling microbial generation of polluting.
Embodiment 6
Difference from Example 5 is, uses 1.5%PHA and 5 μ g/L canavalines (ConA) simultaneously in the aqueous solution that improves artificial seawater, and it is high by 5～10% that biomass improves nutrient solution than embodiment 5.
Embodiment 7
Difference from Example 1 is, chooses the porous microcarrier of gelatin+chitosan Composite Preparation, inoculation condition 8g/L (improvement artificial seawater), and the sponge cell can carry out normal growth.
The preparation reference literature Li KG of the porous microcarrier of gelatin+chitosan Composite Preparation, WangY, Miao ZC, Xu DY, Tang YF, Feng MF.Chitosan/gelatin compositemicrocarrier for hepatocyte culture.Biotechnology Letters, 26:879-883,2004.

Claims (7)

1. the microcarrier three-dimensional culture method of a sponge cell is characterized in that:
1) inoculation microcarrier and sponge cell are cultivated in improving artificial seawater; The solute composition is in the aqueous solution of improvement artificial seawater: Na +300～500mM, SO 4 2-5.0～10.0mM, HCO 3 -0.1～0.5mM, K +5.0～15.0mM, Tris HCl 10.0～30.0mM, Cl -300～500mM, Mg 2+20～60mM, Ca 2+5.0～20mM, SiO 3 2-5～150 μ M, Fe 3+30～120 μ M, Zn 2+0.5～4 μ M, C 6H 5O 7 3-30～120 μ M, pH 7.6～8.2, and the weight concentration of PHA is 1.5～2.5%;
2) after 5～8 days, in above-mentioned improvement artificial seawater, add organic carbon source, organic nitrogen source and vitamins C in the sponge cell cultures, make their concentration be respectively 5～20 μ M, form new nutrient solution system;
3) in step 2) afterwards, changed 50～80% of nutrient solution volume every 2～3 days, replace and add organic carbon source and nitrogenous source makes its ultimate density reach 20～50 μ M with improved artificial seawater, add vitamins C and make its ultimate density reach 10～20 μ M, the sponge cell can carry out normal growth with this understanding.
2. according to the microcarrier three-dimensional culture method of the described sponge cell of claim 1, it is characterized in that: described organic carbon source is glucose, sorbyl alcohol and/or Sodium.alpha.-ketopropionate; Organic nitrogen source is glycine, l-asparagine and/or glutamine.
3. according to the microcarrier three-dimensional culture method of the described sponge cell of claim 1, it is characterized in that: described microcarrier can be selected solid microcarrier or porous microcarrier, the inoculation condition of solid microcarrier is every liter and improves artificial seawater 5～20g that the inoculation condition of porous microcarrier is every liter and improves artificial seawater 2～10g.
4. according to the microcarrier three-dimensional culture method of the described sponge cell of claim 1, it is characterized in that: described sponge cell inoculation scope is 1 * 10 6～9 * 10 7Cells/ml.
5. according to the microcarrier three-dimensional culture method of the described sponge cell of claim 1, it is characterized in that: also can add DMEM or RPMI 1640 in described organic carbon source and nitrogenous source interpolation, making its volumetric concentration in nutrient solution is 0.01%.
6. according to the microcarrier three-dimensional culture method of the described sponge cell of claim 1, it is characterized in that: also can add canavaline in the aqueous solution of described improvement artificial seawater, its addition is every liter of nutrient solution 5～15 μ g/L.
7. according to the microcarrier three-dimensional culture method of the described sponge cell of claim 1, it is characterized in that: the solute composition is in the aqueous solution of described improvement artificial seawater: Na +350～400mM, SO 4 2-7.0mM, HCO 3 -0.2mM, K +10.0mM, Tris HCl 20.0mM, Cl -350～400mM, Mg 2+50mM, Ca 2+10.0mM, SiO 3 2-5～150 μ M, Fe 3+40～80 μ M, Zn 2+0.5～4 μ M, C 6H 5O 7 3-40～50 μ M, pH 7.6～8.2, and the weight concentration of PHA is 1.5～2.5%.
CNB200410020708XA 2004-06-09 2004-06-09 3D culture method for micro carrier of sponge cell Expired - Fee Related CN1332024C (en)

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PT2726600T (en) * 2011-07-01 2017-04-24 Amgen Inc Mammalian cell culture
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Citations (2)

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Publication number Priority date Publication date Assignee Title
US6054317A (en) * 1995-11-22 2000-04-25 Mcmahon; Peter System for the cell culture and cryopreservation of marine invertebrates
CN1490399A (en) * 2002-10-18 2004-04-21 中国科学院大连化学物理研究所 Method for forming block from divided sponge organs

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054317A (en) * 1995-11-22 2000-04-25 Mcmahon; Peter System for the cell culture and cryopreservation of marine invertebrates
CN1490399A (en) * 2002-10-18 2004-04-21 中国科学院大连化学物理研究所 Method for forming block from divided sponge organs

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