CN104694454A - Micro-carrier for cell culture as well as preparation method and application of micro-carrier - Google Patents
Micro-carrier for cell culture as well as preparation method and application of micro-carrier Download PDFInfo
- Publication number
- CN104694454A CN104694454A CN201510090678.8A CN201510090678A CN104694454A CN 104694454 A CN104694454 A CN 104694454A CN 201510090678 A CN201510090678 A CN 201510090678A CN 104694454 A CN104694454 A CN 104694454A
- Authority
- CN
- China
- Prior art keywords
- protein
- polypeptide
- microballoon
- microcarrier
- reaction
- 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.)
- Granted
Links
Landscapes
- Manufacturing Of Micro-Capsules (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to a micro-carrier for cell culture as well as a preparation method and application of the micro-carrier. The carrier comprises positive charge and a protein/peptide coating. The preparation method of the micro-carrier comprises the following steps: (1) carrying out chemical crosslinking on matrix of the micro-carrier and positive charge modifier; and (2) crosslinking matrix microspheres with positive charge obtained from the step (1) and protein/peptide. The invention further provides application of the micro-carrier in cell culture. The micro-carrier is capable of limiting the thickness of the protein/peptide coating, so that the micro-carrier can fully play a role of the protein/peptide, does not completely shield the charge, has the advantage that positive charge is easily adhered to cells, and also has the functions of promoting the protein/peptide and regulating and controlling cell growth, so as to play dual roles of the charge and the protein/peptide and effectively regulate and control the amount; and the micro-carrier has obvious advantages in cell culture and tissue engineering.
Description
Technical field
The present invention relates to bioengineering field, be specifically related to a kind of microcarrier for cell cultures and its preparation method and application.
Background technology
Zooblast be the pharmaceutical grade proteins such as vaccine by main production carrier.In the production process of vaccine, the cultivation of zooblast and expression determine output and the production efficiency of vaccine.In the large scale culturing technology of zooblast, microcarrier culture method has multiple advantage: its specific surface area is large, and the cell yield of unit volume nutrient solution is high; Employing even suspension is cultivated, without nutrition or product gradient; Be easy to the growing state observing micro-carrier surface; Cell harvesting process is relatively simple, and labour intensity is little; Substratum utilization ratio is high, and floor space is little and amplify easy etc., is the most rising a kind of training mode of generally acknowledging at present.Due to the attention to this technology, developed a large amount of microcarrier abroad, part commercialization, its main substrate material has sephadex, Mierocrystalline cellulose, protein, high molecular synthetic material, inorganic glass materials and liquid film microcarrier etc.Take sephadex as the microcarrier of substrate material be use a widest class at present, familiar Cytodex series (GE company) is exactly this kind of microcarrier, wherein Cytodex1 (i.e. DEAE-sephadex) is the widest a kind of microcarrier of current applications, with sephadex microballoon for matrix, then the microcarrier with positive charge that in coupling prepared by DEAE.Also have Cytodex3, it is connected on cross-linked dextran substrate by the mode of chemical coupling one deck denatured collagen (from pigskin type i collagen).
The substrate material of the performance of microcarrier and structure, especially microcarrier, wetting ability, surface chemical structure, charge property and surface shape, matrix structure etc. have material impact to the attaching of cell, growth and propagation.In the practical application of cell cultures microcarrier, it is found that microcarrier with positive charge is compared with protein/polypeptide coating microcarrier, the former cell attachment is faster, and the latter's cell growth rate is higher, if the two can be combined, to be the microcarrier that performance is more superior, this be low for some adhesive powers, more crucial to the more sensitive clone of charge ratio again.Interaction between the cell-vector that the low needs of adhesive power are stronger, to promote the attaching of cell on microcarrier, again limit electric density to charge sensitive should be not too high, otherwise the growth of meeting T suppression cell under " toxicity " effect of electric charge, electric charge-albumen or electric charge-polypeptide dual function microcarrier then can solve this problem.At present, the research of associated charge and protein/polypeptide dual function microcarrier report is also few, general use charged material and protein/polypeptide blended after, then form film or be prepared into microballoon.Its charged material is generally chitosan and urethane, and this microcarrier is that substrate material and the blended of protein/polypeptide are prepared from, or first to make after microballoon coupling protein/polypeptide again; Which kind of method protein/polypeptide is all dispersed in microballoon inside due to molecular diffusion effect.In fact, the effect of cell and microcarrier mainly betides the surface of microcarrier, the protein/polypeptide being scattered in microballoon inside can not play too large effect, especially some polypeptide is expensive, enter microballoon in a large number inner and do not play a role and can cause huge waste, therefore product cost improves, and limits the widespread use of electric charge-protein/polypeptide dual function microcarrier.
Summary of the invention
The object of the present invention is to provide a kind of protein/polypeptide coating thickness controlled and there is microcarrier for cell cultures of positive charge and protein/polypeptide dual function and its preparation method and application.
On the one hand, the invention provides a kind of microcarrier for cell cultures, described microcarrier is with positive charge and protein/polypeptide coating.
Preferably, the thickness of described protein/polypeptide coating is controlled;
Preferably, the thickness of described protein/polypeptide coating is 5 ~ 50% of microcarrier matrix radius, such as, can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of microcarrier matrix radius; Be preferably 10 ~ 30%.In the thickness limited, microcarrier can give full play to the effect of albumen, polypeptide, can not cover electric charge completely again, can play the dual function of electric charge and albumen, polypeptide.
The thickness of protein/polypeptide uses laser confocal microscope to measure, preferred use fluorescein isothiocyanate (FITC) is fluorescent indicator, because FITC can be combined with protein/polypeptide easily, and be not adsorbed onto on polysaccharide microsphere, therefore green fluorescence bright on protein/polypeptide coating can be observed, to determine the thickness of protein/polypeptide layer.
Preferably, described albumen is a kind of in fibronectin, vitronectin, collagen protein, ln, elastin, chondronectin or gelatin or the wherein mixture of at least two kinds; Described gelatin is a kind of in pig gelatin, isinglass or Ox horn Glue or the wherein mixture of at least two kinds, the mixture of the mixture of the mixture of such as pig gelatin or Ox horn Glue or pig gelatin and isinglass, isinglass and Ox horn Glue or pig gelatin, isinglass and Ox horn Glue three;
Preferably, the molecular weight of described albumen is 3,000 ~ 50,000, and can be such as 3,000,4,000,5,000,6,000,7,000,8,000,9,000,10,000,20,000,30,000,40,000 or 50,000; Be preferably 5,000 ~ 50,000, further preferably 5,000 ~ 20,000;
Preferably, described polypeptide is arginine-glycine-aspartic acid, Tyrosine-Isoleucine-glycine-serine-arginine, Isoleucine-lysine-val-ala-dehydroretinol, poly-lysine, polyornithine or the albumen containing aforementioned polypeptides fragment;
Preferably, the electric density of described microcarrier is 0.1 ~ 10mmol/g dry bulb, such as, can be 0.1mmol/g dry bulb, 0.2mmol/g dry bulb, 0.3mmol/g dry bulb, 0.4mmol/g dry bulb, 0.5mmol/g dry bulb, 0.6mmol/g dry bulb, 0.7mmol/g dry bulb, 0.8mmol/g dry bulb, 0.9mmol/g dry bulb, 1mmol/g dry bulb, 2mmol/g dry bulb, 3mmol/g dry bulb, 4mmol/g dry bulb, 5mmol/g dry bulb, 6mmol/g dry bulb, 7mmol/g dry bulb, 8mmol/g dry bulb, 9mmol/g dry bulb or 10mmol/g dry bulb; Be preferably 0.5 ~ 5mmol/g dry bulb, further preferred 0.8 ~ 3mmol/g dry bulb; Matrix microspheres coupling positive charge or the microballoon prepared with the material of electric charge by self, after further coupling protein and polypeptide, due to the bridging effect of protein polypeptide, original electric density can decline to some extent, and therefore its final electric density controls at 0.1 ~ 10mmol/g dry bulb;
Electric density is characterized by the complete exchange capacity measuring described dual function microcarrier, and concrete grammar is with reference to CN 102732475 A.
Preferably, the matrix of described microcarrier is natural polymer or synthesis polymer, described natural polymer is polyose, is preferably Rhizoma amorphophalli glucomannan, dextran, agarose, alginates, chitosan or Mierocrystalline cellulose, more preferably Rhizoma amorphophalli glucomannan or dextran; Described synthesis polymer is polystyrene, urethane or polyethylene;
Preferably, the particle diameter of described microcarrier is 50 ~ 800 μm, such as, can be 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm or 800 μm; Be preferably 100 ~ 300 μm.
On the other hand, the invention provides the preparation method of microcarrier as described in relation to the first aspect, comprise the steps:
(1) by the matrix of microcarrier and the chemically crosslinked of positive charge modifier;
(2) the positively charged matrix microspheres that step (1) obtains is cross-linked with protein/polypeptide again.
Preferably, step (1) comprises the steps:
A) in matrix microspheres, add activator, carry out priming reaction, obtain activating microballoon;
B), after step a) has been reacted, wash away unreacted activator, drain;
C) by step b) system of having reacted adds Na
2cO
3-Na
2hCO
3in damping fluid, then the microballoon after adding positive charge modifier and activating carries out crosslinking reaction;
D) step c) reacted after, wash away unreacted material, obtain positively charged matrix microspheres;
Preferably, step (2) comprises the steps:
E) add cleaning the positively charged matrix microspheres after draining together with protein/polypeptide in damping fluid, stirring reaction;
F) condensing steps e) reacted product, cleaning is drained;
H) rush liquid cleaning with deionized water is gentle successively, drain, obtain the positively charged microcarrier of protein/polypeptide coating;
Optionally, step (2) can also adopt following steps:
2-1: the activation of positively charged matrix microspheres;
2-2: the positively charged matrix microspheres after activation and the combination of protein/polypeptide;
Wherein, step 2-1 comprises the steps:
I) drain clean for positively charged matrix microspheres;
J) in step I) add activator, stirring reaction in the matrix microspheres of gained;
K) step j) terminate after, gained feed liquid is drained, again drains after cleaning, obtain the positively charged matrix microspheres after activation;
Wherein, step 2-2 comprises the steps:
L) drain clean for positively charged matrix microspheres;
M) by step l) gained matrix microspheres, and protein/polypeptide adds in damping fluid, stirring reaction;
N) condensing steps m) products therefrom, cleans and after draining, adds the linking agent of damping fluid and protein/polypeptide molecule, constant temperature stirring reaction;
O) step n is drained) gained feed liquid, cleaning is also drained again, obtains the positively charged microcarrier of protein/polypeptide coating.
When positively charged matrix microspheres and protein/polypeptide generation crosslinking reaction, the pH value of described reaction system is higher than the iso-electric point of protein/polypeptide.
Preferably, the linking agent of described activator and protein/polypeptide molecule is the compound replaced with bifunctional or polyfunctional group; Described functional group is selected from halogen, amido, epoxy group(ing), the glycidyl ether of replacement, guanidine radicals, alkylene or carboxyl; The compound that described bifunctional or polyfunctional group replace is selected from the alkane of halogen and epoxy group(ing) replacement, the glycidyl ether of alkenyl substituted or the amino alkene replaced; Be preferably epoxy chloropropane, epoxy chloropropane-polyol derivative, butane diepoxide or BDO ether; The linking agent of described protein/polypeptide molecule has the functional group can reacted with protein/polypeptide; Bifunctional on described linking agent or polyfunctional group are connected with microballoon, positive charge modifier or protein/polypeptide coating thing respectively, or bifunctional on described linking agent or polyfunctional group are connected with microballoon, modifier or coating thing respectively after overactivation.
Preferably, step a) described in the consumption of activator be 0.1 ~ 100mmol/g microballoon, it can be such as 0.1mmol/g microballoon, 0.5mmol/g microballoon, 1mmol/g microballoon, 2mmol/g microballoon, 3mmol/g microballoon, 4mmol/g microballoon, 5mmol/g microballoon, 6mmol/g microballoon, 7mmol/g microballoon, 8mmol/g microballoon, 9mmol/g microballoon, 10mmol/g microballoon, 20mmol/g microballoon, 30mmol/g microballoon, 40mmol/g microballoon, 50mmol/g microballoon, 60mmol/g microballoon, 70mmol/g microballoon, 80mmol/g microballoon, 90mmol/g microballoon or 100mmol/g microballoon, be preferably 1 ~ 50mmol/g microballoon, described temperature of reaction is 15 ~ 80 DEG C, such as, can be 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C or 80 DEG C, is preferably 35 ~ 50 DEG C, reaction times is 0.5 ~ 24h, such as, can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 12h or 24h, is preferably 2 ~ 8h,
Preferably, step c) described in positive charge modifier be amine or amine salt, be preferably NH
2r1, NHR1R2, NR1R2R3, R4-CO-NH-R5 or its customary salt, wherein R1, R2, R3, R4, R5 is selected from the alkane of substituted or unsubstituted C1 ~ 15 straight or branched respectively, substituted or unsubstituted C1 ~ 15 straight or branched alkene, the more preferably alkane of substituted or unsubstituted C2 ~ 12 straight or branched, substituted or unsubstituted C2 ~ 12 straight or branched alkene, be more preferably 2-diethylin monochloroethane hydrochloride, hexanediamine, diethylamine, trimethylammonium thanomin, N-[3-(dimethylamino) propyl group] Methacrylamide, polymine, Tri N-Propyl Amine, N-heptyl amice, N-amino dodecane or thanomin, described substituting group is selected from amino, hydroxyl or halogen, described positive charge modifier also comprises poly positive charge polymer, is preferably chitosan, polyetherimide or oligochitosan, crosslinking reaction temperature is 15 ~ 80 DEG C, such as, can be 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C or 80 DEG C, cross-linking reaction time is 0.5 ~ 24h, such as, can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 12h or 24h,
Preferably, step e) described in positively charged matrix microspheres and the mass ratio of protein/polypeptide be 2:1 ~ 100:1, can be such as 2:1,3:1,4:1,5:1,10:1,50:1 or 100:1, be preferably 3:1 ~ 10:1; Described temperature of reaction is 15 ~ 80 DEG C, such as, can be 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C or 80 DEG C, is preferably 35 ~ 50 DEG C; Reaction times is 0.5 ~ 24h, such as, can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 12h or 24h, is preferably 2 ~ 8h; Mixing speed is 10 ~ 300rpm, such as, can be 10rpm, 50rpm, 100rpm, 150rpm, 200rpm, 250rpm or 300rpm;
Preferably, step f) and step h) between can also comprise step g), step g) for step f) complete after system in add damping fluid and molecule crosslinked dose of protein/polypeptide, constant temperature stirring reaction, drain gained feed liquid after reaction terminates; Molecule crosslinked dose of described protein/polypeptide has the functional group can reacted with protein/polypeptide; The consumption that protein/polypeptide is molecule crosslinked dose is 0.1 ~ 100mmol/g microballoon, can be such as 0.1mmol/g microballoon, 1mmol/g microballoon, 10mmol/g microballoon, 20mmol/g microballoon, 30mmol/g microballoon, 40mmol/g microballoon, 50mmol/g microballoon, 60mmol/g microballoon, 70mmol/g microballoon, 80mmol/g microballoon, 90mmol/g microballoon or 100mmol/g microballoon, be preferably 1 ~ 50mmol/g microballoon; Described temperature of reaction is 15 ~ 80 DEG C, such as, can be 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C or 80 DEG C, is preferably 35 ~ 50 DEG C; Reaction times is 0.5 ~ 24h, such as, can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 12h or 24h, is preferably 2 ~ 8h; Mixing speed is 10 ~ 300rpm, such as, can be 10rpm, 50rpm, 100rpm, 150rpm, 200rpm, 250rpm or 300rpm;
Preferably, at described step j) in, to step I) complete after gained matrix microspheres in can also add oxidation inhibitor, stirring reaction; The consumption of described linking agent is 0.1 ~ 100mmol/g microballoon, can be such as 0.1mmol/g microballoon, 1mmol/g microballoon, 10mmol/g microballoon, 20mmol/g microballoon, 30mmol/g microballoon, 40mmol/g microballoon, 50mmol/g microballoon, 60mmol/g microballoon, 70mmol/g microballoon, 80mmol/g microballoon, 90mmol/g microballoon or 100mmol/g microballoon, preferably 1 ~ 50mmol/g microballoon; Reaction times is 0.5 ~ 24h, such as, can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 12h or 24h, is preferably 2 ~ 8h; Mixing speed is 10 ~ 300rpm, such as, can be 10rpm, 50rpm, 100rpm, 150rpm, 200rpm, 250rpm or 300rpm;
Preferably, described method can also comprise being cross-linked between protein/polypeptide molecule, cross-linking reagent used is dialdehyde base class, bis-epoxy class, diimine class, nitrine quasi-molecule or the wherein mixture of at least two kinds, be preferably mda, suceinic aldehyde, carbodiimide, vulcabond, genipin, glutaraldehyde or nitrine diphenylphosphine or the wherein mixture of at least two kinds, more preferably suceinic aldehyde, carbodiimide, vulcabond or the wherein mixture of at least two kinds; The consumption of cross-linking reagent is 0.1 ~ 100mmol/g microballoon, can be such as 0.1mmol/g microballoon, 1mmol/g microballoon, 10mmol/g microballoon, 20mmol/g microballoon, 30mmol/g microballoon, 40mmol/g microballoon, 50mmol/g microballoon, 60mmol/g microballoon, 70mmol/g microballoon, 80mmol/g microballoon, 90mmol/g microballoon or 100mmol/g microballoon, be preferably 1 ~ 50mmol/g microballoon, more preferably 1 ~ 10mmol/g microballoon; Crosslinking reaction temperature is 15 ~ 65 DEG C, such as, can be 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C or 65 DEG C; Cross-linking reaction time is 0.5 ~ 24h, such as, can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 12h or 24h.
Provided by the invention to have positive charge with the microcarrier of protein/polypeptide dual function be connected positively charged modifier by the microballoon method of chemically crosslinked, more obtained with protein/polypeptide in chemical crosslink technique coupling further; But for itself having the carrier of positive charge, can direct coupling protein/polypeptide.
The chemically crosslinked of described matrix microspheres and positive charge modifier comprises:
(1) matrix microspheres elder generation and linking agent reaction bonded, then reacted by linking agent and positive charge modifier, thus matrix microspheres is connected with modifier;
(2) after the linking agent reaction bonded that matrix microspheres is first not activated with one end, functional group not activated on reactivate linking agent, the functional group after activation and positive charge modifier react, thus matrix microspheres is connected with modifier;
(3) first linking agent is connected with positive charge modifier, then by the functional group on linking agent and matrix microspheres reaction forming.
Except chemical conjugation methods, protein/polypeptide also can on the microballoon of physical adsorption positive charge in coupling.Compared with the method for carrying out coating with physical adsorption, the microcarrier that chemical coupling obtains, its protein/polypeptide stability is better.No matter be physical method or chemical process, when coupling protein/polypeptide, the pH value of solution must be adjusted to more than the iso-electric point of protein/polypeptide, make protein/polypeptide electronegative, adjusted the coating thickness of protein/polypeptide by the comprehensive action of electrostatic force and adsorptive power.The positive charge density of carrier is higher, and protein/polypeptide layer is thinner; The pH of reaction system differs larger with the iso-electric point of protein/polypeptide, and coating thickness is thinner; Therefore, the thickness of protein/polypeptide layer can be adjusted as the case may be.
The third aspect, the invention provides the application of microcarrier in cell cultures as described in relation to the first aspect.
Compared with prior art, the present invention at least has following beneficial effect:
Microcarrier of the present invention can limit the thickness of protein/polypeptide coating, make microcarrier can give full play to the effect of protein/polypeptide, electric charge can not be covered completely again, make it both have advantage that positive charge is easy to adherent cell, there is again the function that protein/polypeptide promotes and regulating cell grows, the dual function of electric charge and protein/polypeptide can be played, and its consumption of Effective Regulation, in cell cultures and organizational project, there is obvious advantage.
Accompanying drawing explanation
Fig. 1 is the laser co-focusing photo of microcarrier prepared by embodiment 13;
Fig. 2 is the laser co-focusing photo of microcarrier prepared by embodiment 16;
Fig. 3 is the laser co-focusing photo of microcarrier prepared by embodiment 18;
Fig. 4 is Rhizoma amorphophalli glucomannan microcarrier Cultivation of Vero result in embodiment 20 (cultivating 144h);
Fig. 5 is that in embodiment 21, Rhizoma amorphophalli glucomannan microcarrier cultivates L929 cell results (cultivating 120h);
Fig. 6 is that in embodiment 22, Rhizoma amorphophalli glucomannan microcarrier cultivates CHO-K1 cell results (cultivating 120h);
Fig. 7 is that in embodiment 23, Rhizoma amorphophalli glucomannan microcarrier cultivates CHO
22h
11cell results (cultivating 160h);
Fig. 8 is dextran microcarrier Cultivation of Vero result in embodiment 24 (cultivating 200h);
Fig. 9 is that in embodiment 25, dextran microcarrier cultivates bhk cell result (cultivating 144h);
Figure 10 is the cell cultures curve control of different microcarrier Cultivation of Vero in embodiment 26;
Figure 11 is the laser co-focusing photo without electric charge collagen type microcarrier prepared by comparative example 2.
Embodiment
Technical scheme of the present invention is further illustrated below by embodiment.Those skilled in the art should understand, described embodiment is only help to understand the present invention, should not be considered as concrete restriction of the present invention.
Embodiment 1
The present embodiment is the coupling of matrix microspheres and positive charge, and step is as follows:
Get 200g Cross-linked Konjac Glucomannan Granules (Konjac Glucomannan Granules is prepared according to Chinese invention patent CN101113180 B), add the NaOH solution 400mL that concentration is 1.2mol/L, and epoxy chloropropane 200mL, in 35 DEG C of isothermal reaction 2h.After having reacted, wash away unreacted epoxy chloropropane with deionized water, after draining, put into the Na of pH=9.5
2cO
3-Na
2hCO
3in damping fluid, add 10.0g diethylamine, 35 DEG C of isothermal reaction 18h.Wash away unreacted material with deionized water after reaction, namely obtain (positively charged) Konjac Glucomannan Granules of amination.
Embodiment 2
The present embodiment is the coupling of matrix microspheres and positive charge, and step is as follows:
Get 200g sephadex microballoon, add the NaOH solution 300mL that concentration is 1.5mol/L, add 20.0g trimethylammonium thanomin, 35 DEG C of isothermal reaction 18h.Wash away unreacted material with deionized water after reaction, namely obtain amination sephadex microballoon.
Embodiment 3
The present embodiment is the coupling of matrix microspheres and positive charge, and step is as follows:
Get 200g agarose microbeads, add the NaOH solution 400mL that concentration is 1.2mol/L, add 12.0g2-diethylin monochloroethane hydrochloride, 35 DEG C of isothermal reaction 18h.Wash away unreacted material with deionized water after reaction, namely obtain the agarose microbeads of amination.
Embodiment 4
The present embodiment is the coupling of matrix microspheres and positive charge, and step is as follows:
Get 100g dextran microspheres, add the NaOH solution 150mL that concentration is 2.0mol/L, and BDO ether 200mL, in 45 DEG C of isothermal reaction 3h.After having reacted, wash away unreacted BDO ether with deionized water, after draining, put into the Na of pH=9.5
2cO
3-Na
2hCO
3in damping fluid, add 45.0g hexanediamine, 40 DEG C of isothermal reaction 18h.Wash away unreacted material with deionized water after reaction, namely obtain the dextran microspheres of amination.
Embodiment 5
The present embodiment is the coupling of matrix microspheres and positive charge, and step is as follows:
Get the chitosan 100g that deacetylation is 87%, be dissolved in the acetic acid solution of 10%, be mixed with the solution that chitosan concentration is 3%.Get 50g chitosan solution, be scattered in 250ml whiteruss under 100rpm, whiteruss contains the Span85 of 2%.Be warming up to 60 DEG C, add 20g glutaraldehyde, crosslinked 24h.Wash away unreacted material with deionized water after reaction, namely obtain the chitosan microball of amination.
The test result of embodiment 1 ~ 5 is as shown in table 1.
Table 1
| Microballoon water content (%) | Electric density (mmol/g dry bulb) | Microspherulite diameter (μm) |
| Embodiment 1 | 95 | 1.2 | 210 |
| Embodiment 2 | 95 | 3.5 | 100 |
| Embodiment 3 | 80 | 1.6 | 220 |
| Embodiment 4 | 86 | 9.6 | 300 |
| Embodiment 5 | 90 | 0.6 | 500 |
Water content is the concentrated expression of microballoon skeletal density, amination degree and degree of crosslinking, and according to other experimental result in the past, the cell cultures effect of microballoon water content in 80 ~ 95% scopes is better.Can tentatively find out from above result, the effect of microballoon culturing cell of the present invention is better.
Embodiment 6
The present embodiment is the combination of amination microballoon and albumen, and step is as follows:
Amination Konjac Glucomannan Granules prepared by Example 1, drains after cleaning.Get this to clean the microballoon 50g that drains and porcine collagen 5.0g and be placed in Erlenmeyer flask, then add the NaHCO that 200mL concentration is 0.1mol/L
3/ Na
2cO
3damping fluid (pH=10.0), constant temperature stirring reaction 5h under 120rpm.With the cold water condensation of 4 DEG C, drain with after PBS buffer solution for cleaning, be placed in Erlenmeyer flask, add 150mLPBS buffered soln, the glutaraldehyde of 25mL concentration 25%, constant temperature stirring reaction 5h under 120rpm, to be transferred to feed liquid after reaction terminates in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, namely obtaining the amination Rhizoma amorphophalli glucomannan microcarrier of collagen coating.
Embodiment 7
The present embodiment is the combination of amination microballoon and albumen, and step is as follows:
Amination agarose microbeads prepared by Example 3, drains after cleaning.Get this to clean the microballoon 50g that drains and Isin glue collagen 0.5g and be placed in Erlenmeyer flask, then add the NaHCO that 150mL concentration is 0.5mol/L
3/ Na
2cO
3damping fluid (pH=10.8), constant temperature stirring reaction 12h under 60rpm.With the cold water condensation of 4 DEG C, drain with after PBS buffer solution for cleaning, be placed in Erlenmeyer flask, add 150mLPBS buffered soln, the glutaraldehyde of 25mL concentration 10%, constant temperature stirring reaction 12h under 100rpm, to be transferred to feed liquid after reaction terminates in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, namely obtaining the amination agarose microcarrier of collagen coating.
Embodiment 8
The present embodiment is the combination of amination microballoon and albumen, and step is as follows:
Chitosan microball prepared by Example 5, drains after cleaning.Get this clean microballoon 5g and IKVAV (Isoleucine-lysine-val-ala-dehydroretinol) 2.5g drained and be placed in Erlenmeyer flask, then add the NaHCO that 15mL concentration is 0.1mol/L
3/ Na
2cO
3damping fluid (pH=10.0), constant temperature stirring reaction 5h under 120rpm.With the cold water condensation of 4 DEG C, drain with after PBS buffer solution for cleaning, be placed in Erlenmeyer flask, add 150mLPBS buffered soln, the glutaraldehyde of 25mL concentration 25%, with constant temperature stirring reaction 5h under 120rpm, to be transferred to feed liquid after reaction terminates in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, namely obtaining the amination chitosan microcarriers of polypeptide coating.
The test result of embodiment 6 ~ 8 is as shown in table 2.Loss of charge can be produced in whole reaction process, matrix microspheres coupling positive charge or the microballoon prepared with the material of electric charge by self, after further coupling protein and polypeptide, due to the bridging effect of protein polypeptide, original electric density also can decline to some extent, microballoon and protein/polypeptide coating thickness also can be caused unstable and produce particle diameter and variation in thickness, therefore final protein/polypeptide coating thickness, electric density and the microspherulite diameter of the microcarrier of amination microballoon and protein binding mode gained are as the criterion with result shown in table 2.
Table 2
| Protein/polypeptide coating thick (μm) | Electric density (mmol/g dry bulb) | Microspherulite diameter (μm) | |
| Embodiment 6 | 30 | 0.3 | 210 |
| Embodiment 7 | 11 | 1.3 | 220 |
| Embodiment 8 | 150 | 0.1 | 500 |
Embodiment 9
The present embodiment is the activation of amination matrix microspheres, and step is as follows:
Amination Konjac Glucomannan Granules prepared by Example 1, drains after being placed in sand core funnel deionized water wash.Take 10g and should clean the Erlenmeyer flask that the microballoon drained is placed in 100mL, add NaOH solution, 40mL1,4-butanediol diglycidyl ether and 20mg sodium borohydride that 20mL concentration is 0.5mol/L, in 120rpm, 65 DEG C of constant temperature stirring reaction 8h.After reaction terminates, feed liquid to be transferred in sand core funnel and to drain, then draining with dehydrated alcohol and washed with de-ionized water successively, namely obtain the Rhizoma amorphophalli glucomannan amination microballoon that epoxy activates.
Embodiment 10
The present embodiment is the activation of amination matrix microspheres, and step is as follows:
Amination dextran microspheres prepared by Example 4, drains after being placed in sand core funnel deionized water wash.Take 10g and should clean the Erlenmeyer flask that the microballoon drained is placed in 100mL, add NaOH solution, 20mL1,4-butanediol diglycidyl ether that 20mL concentration is 0.1mol/L, in 100rpm, 80 DEG C of constant temperature stirring reaction 12h.After reaction terminates, feed liquid to be transferred in sand core funnel and to drain, then draining with dehydrated alcohol and washed with de-ionized water successively, namely obtain the dextran amination microballoon that epoxy activates.
Embodiment 11
The present embodiment is the activation of amination matrix microspheres, and step is as follows:
Amination dextran microspheres prepared by Example 4, drains after being placed in sand core funnel deionized water wash.Take 20g and should clean the Erlenmeyer flask that the microballoon drained is placed in 100mL, add NaOH solution, 15mL1,4-butanediol diglycidyl ether that 50mL concentration is 0.9mol/L, in 120rpm, 65 DEG C of constant temperature stirring reaction 8h.After reaction terminates, feed liquid to be transferred in sand core funnel and to drain, then draining with dehydrated alcohol and washed with de-ionized water successively, namely obtain the dextran amination microballoon that epoxy activates.
Embodiment 12
The present embodiment is the activation of amination matrix microspheres, and step is as follows:
Activation chitosan microball prepared by embodiment 5, drains after being placed in sand core funnel deionized water wash.Take this and clean the microballoon 50g drained, add NaOH solution, 10mL1,4-butanediol diglycidyl ether that 100mL concentration is 0.3mol/L, in 120rpm, 65 DEG C of constant temperature stirring reaction 8h.After reaction terminates, feed liquid to be transferred in sand core funnel and to drain, then draining with dehydrated alcohol and washed with de-ionized water successively, namely obtain the chitosan amination microballoon that epoxy activates.
Embodiment 13
The present embodiment is positively charged matrix microspheres after activation and the combination of protein/polypeptide, and step is as follows:
The Rhizoma amorphophalli glucomannan amination microballoon of epoxy activation prepared by Example 9, cleans and drains.Take this to clean the microballoon 50g that drains and porcine collagen 5g and be placed in Erlenmeyer flask, add the NaHCO that 150mL concentration is 0.1mol/L
3/ Na
2cO
3damping fluid (pH=10.0), constant temperature stirring reaction 5h under 120rpm.With the cold water condensation of 4 DEG C, then with the cleaning of PBS buffered soln, drain and be placed in Erlenmeyer flask, add 120mLPBS damping fluid and 25mL concentration is the glutaraldehyde of 25%, constant temperature stirring reaction 5h under 120rpm, after reaction terminates, to be transferred to feed liquid in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, obtaining the amination Rhizoma amorphophalli glucomannan microcarrier of collagen coating.
This microcarrier, after damping fluid rinses repeatedly, not to detect collagen protein in supernatant liquor.Cell cultures is observed after repeating experiment three times under laser confocal microscope, and find that the thickness of egg white layer considerable change does not occur, meanwhile, three times cell cultures result is close.
Embodiment 14
The present embodiment is positively charged matrix microspheres after activation and the combination of protein/polypeptide, and step is as follows:
The Rhizoma amorphophalli glucomannan amination microballoon of epoxy activation prepared by Example 9, cleans and drains.Take this to clean the microballoon 50g that drains and porcine collagen 0.5g and be placed in Erlenmeyer flask, add the NaHCO that 150mL concentration is 0.1mol/L
3/ Na
2cO
3damping fluid (pH=10.0), constant temperature stirring reaction 5h under 120rpm.With the cold water condensation of 4 DEG C, then with the cleaning of PBS buffered soln, drain and be placed in Erlenmeyer flask, add 120mLPBS damping fluid and 2.5mL concentration is the glutaraldehyde of 25%, constant temperature stirring reaction 5h under 120rpm, after reaction terminates, to be transferred to feed liquid in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, obtaining the amination Rhizoma amorphophalli glucomannan microcarrier of collagen coating.
Embodiment 15
The present embodiment is positively charged matrix microspheres after activation and the combination of protein/polypeptide, and step is as follows:
The Rhizoma amorphophalli glucomannan amination microballoon of epoxy activation prepared by Example 9, cleans and drains.Take this to clean the microballoon 50g that drains and porcine collagen 15g and be placed in Erlenmeyer flask, add the NaHCO that 150mL concentration is 0.1mol/L
3/ Na
2cO
3damping fluid (pH=9.0), constant temperature stirring reaction 5h under 120rpm.With the cold water condensation of 4 DEG C, then with the cleaning of PBS buffered soln, drain and be placed in Erlenmeyer flask, add 120mLPBS damping fluid and 2.5mL concentration is the glutaraldehyde of 25%, constant temperature stirring reaction 5h under 120rpm, after reaction terminates, to be transferred to feed liquid in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, obtaining the amination Rhizoma amorphophalli glucomannan microcarrier of collagen coating.
Embodiment 16
The present embodiment is positively charged matrix microspheres after activation and the combination of protein/polypeptide, and step is as follows:
The dextran amination microballoon of epoxy activation prepared by Example 10, cleans and drains.Take this to clean the microballoon 5g that drains and porcine collagen 0.5g and be placed in Erlenmeyer flask, add the NaHCO that 15mL concentration is 0.1mol/L
3/ Na
2cO
3damping fluid (pH=12.0), constant temperature stirring reaction 5h under 120rpm.With the cold water condensation of 4 DEG C, then with the cleaning of PBS buffered soln, drain and be placed in Erlenmeyer flask, add 15mLPBS damping fluid and 2.5mL concentration is the glutaraldehyde of 25%, constant temperature stirring reaction 5h under 120rpm, after reaction terminates, to be transferred to feed liquid in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, obtaining the amination Rhizoma amorphophalli glucomannan microcarrier of collagen coating.
Embodiment 17
The present embodiment is positively charged matrix microspheres after activation and the combination of protein/polypeptide, and step is as follows:
The dextran amination microballoon of epoxy activation prepared by Example 10, cleans and drains.Take this to clean the microballoon 5g that drains and porcine collagen 2.4g and be placed in Erlenmeyer flask, add the NaHCO that 15mL concentration is 0.1mol/L
3/ Na
2cO
3damping fluid (pH=9.5), constant temperature stirring reaction 5h under 120rpm.With the cold water condensation of 4 DEG C, then with the cleaning of PBS buffered soln, drain and be placed in Erlenmeyer flask, add 15mLPBS damping fluid and 2.5mL concentration is the glutaraldehyde of 25%, constant temperature stirring reaction 5h under 120rpm, after reaction terminates, to be transferred to feed liquid in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, obtaining the amination Rhizoma amorphophalli glucomannan microcarrier of collagen coating.
Embodiment 18
The present embodiment is positively charged matrix microspheres after activation and the combination of protein/polypeptide, and step is as follows:
The dextran amination microballoon of epoxy activation prepared by Example 11, cleans and drains.Take this to clean the microballoon 5g that drains and Fibronectin 0.5g and be placed in Erlenmeyer flask, add the NaHCO that 15mL concentration is 0.1mol/L
3/ Na
2cO
3damping fluid (pH=10.0), constant temperature stirring reaction 5h under 120rpm.With the cold water condensation of 4 DEG C, then with the cleaning of PBS buffered soln, drain and be placed in Erlenmeyer flask, add 15mLPBS damping fluid and 2.5mL concentration is the glutaraldehyde of 25%, constant temperature stirring reaction 5h under 120rpm, after reaction terminates, to be transferred to feed liquid in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, obtaining the amination dextran microcarrier of collagen coating.
Embodiment 19
The present embodiment is positively charged matrix microspheres after activation and the combination of protein/polypeptide, and step is as follows:
Chitosan amination microballoon after activation prepared by Example 12, cleans and drains.Take this clean microballoon 5g and IKVAV (Isoleucine-lysine-val-ala-dehydroretinol) 0.2g drained and be placed in Erlenmeyer flask, add the NaHCO that 15mL concentration is 0.1mol/L
3/ Na
2cO
3damping fluid (pH=10.5), constant temperature stirring reaction 5h under 120rpm.With the cold water condensation of 4 DEG C, then with the cleaning of PBS buffered soln, drain and be placed in Erlenmeyer flask, add 15mLPBS damping fluid and 2.5mL concentration is the glutaraldehyde of 25%, constant temperature stirring reaction 5h under 120rpm, after reaction terminates, to be transferred to feed liquid in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, obtaining the amination chitosan microcarriers of polypeptide coating.
The test result of embodiment 13 ~ 19 is as shown in table 3.Loss of charge can be produced in whole reaction process, matrix microspheres coupling positive charge or the microballoon prepared with the material of electric charge by self, after further coupling protein and polypeptide, due to the bridging effect of protein polypeptide, original electric density also can decline to some extent, also can cause the change of microballoon and protein/polypeptide coating thickness instability and thickness and particle diameter, therefore with first activation zone positive electricity matrix microspheres, the final protein/polypeptide coating thickness of the microcarrier of the mode gained be combined with protein/polypeptide again, electric density and microspherulite diameter are as the criterion with result shown in table 3.
Table 3
| Protein/polypeptide coating thickness (μm) | Electric density (mmol/g dry bulb) | Microspherulite diameter (μm) | |
| Embodiment 13 | 30 | 0.8 | 210 |
| Embodiment 14 | 5 | 1.2 | 210 |
| Embodiment 15 | 50 | 0.3 | 210 |
| Embodiment 16 | 60 | 2.7 | 300 |
| Embodiment 17 | 75 | 2.2 | 300 |
| Embodiment 18 | 10 | 3.5 | 300 |
| Embodiment 19 | 150 | 0.1 | 500 |
Embodiment 20
Rhizoma amorphophalli glucomannan microcarrier Cultivation of Vero prepared by Example 13, cell culture condition is: cell-seeding-density 1.5 ~ 2.0 × 10
5cells/mL, microcarrier density 2.0 ~ 2.5g/L, cultivation final volume 100ml, nutrient solution are DMEM/M199 (1:1)+10% foetal calf serum, and cultivate 168 hours, cell density can reach 1.0 ~ 1.2 × 10
6cells/mL.
Embodiment 21
Rhizoma amorphophalli glucomannan microcarrier prepared by Example 13 cultivates L929 cell, and cell culture condition is: cell-seeding-density 2.5 ~ 3.0 × 10
5cells/mL, microcarrier density 5g/L, volume of culture 20mL, cultivate 168 hours, cell maximum density reaches 1.0 ~ 1.5 × 10
6cells/mL.
Embodiment 22
Rhizoma amorphophalli glucomannan microcarrier prepared by Example 13 cultivates CHO-K1 cell, and cell culture condition is: cell-seeding-density 2.0 ~ 2.5 × 10
5cells/mL, Microsphere Density 2.5 ~ 3.0g/L, cultivation final volume 100mL, nutrient solution are Ham F-12+5% foetal calf serum, and cultivate 144 hours, cell maximum density can reach 1.0 ~ 1.5 × 10
6cells/mL.
Embodiment 23
The CHO of Rhizoma amorphophalli glucomannan microcarrier culturing gene restructuring prepared by Example 13
22h
11cell, the gene containing encoding human source chimeric antibody in this cell.Cell culture condition is: cell-seeding-density 2.0 ~ 2.5 × 10
5cells/mL, microcarrier density is 2.5 ~ 3.0g/L, volume of culture is 100mL, and nutrient solution is DMEM/F-12 (1:1)+10% foetal calf serum, and cultivate 172 hours, cell density reaches 1.0 ~ 1.2 × 10
6cells/mL.
Embodiment 24
Dextran microcarrier Cultivation of Vero prepared by Example 16, cell culture condition is: cell-seeding-density 1.5 ~ 2.0 × 10
5cells/mL, Microsphere Density 2.0 ~ 2.5g/L, nutrient solution final volume 100mL, nutrient solution are DMEM/M199 (1:1)+5% foetal calf serum, and cultivate 216 hours, cell maximum density can reach 1.5 ~ 1.8 × 10
6cells/mL.
Embodiment 25
Dextran microcarrier prepared by Example 17 cultivates bhk cell, and cell culture condition is: cell-seeding-density 2.0 ~ 2.5 × 10
5cells/mL, Microsphere Density 4g/L, cultivation final volume 80mL, nutrient solution M199+5%FBS, cultivate 168 hours, cell density reaches 3.0 ~ 3.5 × 10
6cells/mL.
Fig. 4 ~ 9 show, and microsphere surface is the cell of growth, and microphotograph can illustrate intuitively, and through the cultivation of certain hour, the growing state on the dual function microcarrier that cell is prepared in the present invention is good.
Embodiment 26
The Vero cell that Rhizoma amorphophalli glucomannan microcarrier prepared by Example 13 is cultivated, cell culture condition is: cell-seeding-density 10 ~ 12 × 10
4cells/mL, microcarrier density 2.0 ~ 2.5g/L, cultivation final volume 100mL, nutrient solution are DMEM/M199 (1:1)+10% foetal calf serum, and cultivate 216 hours, cell density can reach 1.40 × 10
6cells/mL, cell growth curves is shown in the curve 1 in accompanying drawing 10.
Comparative example 1
Get the Cross-linked Konjac Glucomannan Granules (Konjac Glucomannan Granules is prepared according to Chinese invention patent CN 101113180 B) of 20.0g.Add the NaOH solution 20mL that concentration is 1.2mol/L, 70 DEG C of isothermal reaction 2h.React the Na putting into pH=9.5
2cO
3-Na
2hCO
3in damping fluid, add 10.0gDEAE, 70 DEG C of isothermal reaction 15h.After having reacted, wash away unreacting substance with deionized water, obtain Rhizoma amorphophalli glucomannan amination microcarrier, the water content recording microcarrier is 91.0%.
Comparative example 2
Get appropriate Cross-linked Konjac Glucomannan Granules (Konjac Glucomannan Granules is prepared according to Chinese invention patent CN101113180 B) and be placed in sand core funnel, to use after deionized water wash and to drain.Take 10g and clean the microballoon drained, be placed in the Erlenmeyer flask of 100mL, add NaOH solution, 40mL1,4-butanediol diglycidyl ether and 20mg sodium borohydride that 20mL concentration is 0.5mol/L, at 120rpm, 65 DEG C of constant temperature stirring reaction 8h.After reaction terminates, feed liquid to be transferred in sand core funnel and to drain, then using dehydrated alcohol and washed with de-ionized water successively, drain, namely obtaining the Rhizoma amorphophalli glucomannan amination microballoon of epoxy activation.
Get the Konjac Glucomannan Granules after appropriate activation, clean and drain.Take cleaning and drain microballoon 5g and gelatin 0.5g is placed in Erlenmeyer flask, adding 15mL concentration is the NaHCO of 0.1mol/L
3/ Na
2cO
3damping fluid (pH=10.0).Constant temperature stirring reaction 5h under 120rpm.With 4 DEG C of cold water condensations, PBS buffered soln cleans, drain as in Erlenmeyer flask, add 15mLPBS buffered soln, 2.5mL25% glutaraldehyde, constant temperature stirring reaction 5h under 120rpm, after reaction terminates, feed liquid to be transferred in sand core funnel and to drain, then clean with deionized water and PBS buffered soln and drain successively, obtaining the Rhizoma amorphophalli glucomannan microcarrier of collagen coating.Gained microcarrier is with after FITC dyeing, and find that albumen is uniformly distributed in microcarrier, result as shown in figure 11.
Meanwhile, get Cultivation of Vero under microcarrier Cytodex1, Cytodex3 and comparative example 1 and comparative example 2 the same terms that GE company produces, obtain cell culture density and be respectively 1.02 × 10
6cells/mL, 1.23 × 10
6cells/mL, 0.90 × 10
6cells/mL, 1.10 × 10
6cells/mL, its cell growth curves is shown in the curve 2,3,4,5 in accompanying drawing 10 respectively.As can be seen from cell growth curves, cultivate curve (curve 1) compared with the cultivation curve (curve 2 ~ 5) of four comparative examples at microcarrier of the present invention, the attaching speed of cell and the cell cultures quantity finally reached all have obvious advantage.
As can be seen from Fig. 1 ~ 3 and Figure 11, the protein/polypeptide coating uniform fold of microcarrier prepared by the present invention is in the surface of matrix microspheres, simultaneously, coating thickness is controlled, and the albumen without electric charge collagen type microcarrier prepared by comparative example 2 has entered the inside of matrix microspheres in a large number, the function of albumen cannot be given full play to, serious waste protein material, with high costs.Visible, microcarrier prepared by the present invention can give full play to the effect of protein/polypeptide, also there is the protein/polypeptide coating of controllable thickness, the synergy of electric charge-protein/polypeptide can be given full play to again, superior performance, make that cell adhesion is fast, growth velocity is high, be especially applicable to that adhesion rate is low, the cultivation of charge sensitive class cell.
Applicant states, the present invention illustrates processing method of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned processing step, does not namely mean that the present invention must rely on above-mentioned processing step and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of ancillary component, the concrete way choice etc. of raw material selected by the present invention, all drops within protection scope of the present invention and open scope.
Claims (10)
1. for a microcarrier for cell cultures, it is characterized in that, described microcarrier has positive charge and protein/polypeptide coating simultaneously.
2. microcarrier according to claim 1, is characterized in that, the thickness of described protein/polypeptide coating is controlled;
Preferably, the thickness of described protein/polypeptide coating is 5 ~ 50% of microcarrier matrix radius, is preferably 10 ~ 30%.
3. microcarrier according to claim 1 and 2, is characterized in that, described albumen is the mixing of any one or at least two kinds in fibronectin, vitronectin, collagen protein, ln, elastin, chondronectin or gelatin; Described gelatin is preferably the mixing of any one or at least two kinds in pig gelatin, isinglass or Ox horn Glue;
Preferably, the molecular weight of described albumen is 3,000 ~ 50,000, be preferably 5,000 ~ 50,000, more preferably 5,000 ~ 20,000;
Preferably, described polypeptide is arginine-glycine-aspartic acid, Tyrosine-Isoleucine-glycine-serine-arginine, Isoleucine-lysine-val-ala-dehydroretinol, poly-lysine, polyornithine or the albumen containing aforementioned polypeptides fragment.
4. the microcarrier according to any one of claims 1 to 3, is characterized in that, the electric density of described microcarrier is 0.1 ~ 10mmol/g dry bulb, is preferably 0.5 ~ 5mmol/g dry bulb, further preferred 0.8 ~ 3mmol/g dry bulb.
5. the microcarrier according to any one of Claims 1 to 4, it is characterized in that, the matrix of described microcarrier is natural polymer or synthesis polymer, described natural polymer is polyose, be preferably Rhizoma amorphophalli glucomannan, dextran, agarose, alginates, chitosan or Mierocrystalline cellulose, more preferably Rhizoma amorphophalli glucomannan or dextran; Described synthesis polymer is polystyrene, urethane or polyethylene;
Preferably, the particle diameter of described microcarrier is 50 ~ 800 μm, is preferably 100 ~ 300 μm.
6. the preparation method of the microcarrier according to any one of Claims 1 to 5, is characterized in that, described method comprises:
(1) by the matrix of microcarrier and the chemically crosslinked of positive charge modifier;
(2) the positively charged matrix microspheres that step (1) obtains is cross-linked with protein/polypeptide again.
7. method according to claim 6, is characterized in that, step (1) comprises the steps:
A) in matrix microspheres, add activator, carry out priming reaction, obtain activating microballoon;
B), after step a) has been reacted, wash away unreacted activator, drain;
C) by step b) system of having reacted adds Na
2cO
3-Na
2hCO
3in damping fluid, then the microballoon after adding positive charge modifier and activating carries out crosslinking reaction;
D) step c) reacted after, wash away unreacted material, obtain positively charged matrix microspheres.
Preferably, step (2) comprises the steps:
E) add cleaning the positively charged matrix microspheres after draining together with protein/polypeptide in damping fluid, stirring reaction;
F) condensing steps e) reacted product, cleaning is drained;
H) rush liquid cleaning with deionized water is gentle successively, drain, obtain the positively charged microcarrier of protein/polypeptide coating;
Optionally, step (2) can also adopt following steps:
2-1: the activation of positively charged matrix microspheres;
2-2: the positively charged matrix microspheres after activation and the combination of protein/polypeptide;
Wherein, step 2-1 comprises the steps:
I) drain clean for positively charged matrix microspheres;
J) in step I) complete after gained matrix microspheres in add activator, stirring reaction;
K) step j) terminate after, gained feed liquid is drained, again drains after cleaning, obtain the positively charged matrix microspheres after activation;
Wherein, step 2-2 comprises the steps:
L) drain clean for positively charged matrix microspheres;
M) by step l) gained matrix microspheres, and protein/polypeptide adds in damping fluid, stirring reaction;
N) condensing steps m) reaction terminates the product of rear gained, and clean and after draining, add the linking agent of damping fluid and protein/polypeptide molecule, constant temperature stirring reaction;
O) step n is drained) gained feed liquid, cleaning is also drained again, obtains the positively charged microcarrier of protein/polypeptide coating.
When positively charged matrix microspheres and protein/polypeptide crosslinking reaction, the pH value of described reaction system is higher than the iso-electric point of protein/polypeptide.
8. the preparation method according to claim 6 or 7, is characterized in that, the linking agent of described activator and protein/polypeptide molecule is the compound replaced with bifunctional or polyfunctional group; Described functional group is selected from halogen, amido, epoxy group(ing), the glycidyl ether of replacement, guanidine radicals, alkylene or carboxyl; The compound that described bifunctional or polyfunctional group replace is selected from the alkane of halogen and epoxy group(ing) replacement, the glycidyl ether of alkenyl substituted or the amino alkene replaced; Be preferably epoxy chloropropane, epoxy chloropropane-polyol derivative, butane diepoxide or BDO ether;
Preferably, step a) described in the consumption of activator be 0.1 ~ 100mmol/g microballoon, be preferably 1 ~ 50mmol/g microballoon; Described temperature of reaction is 15 ~ 80 DEG C, is preferably 35 ~ 50 DEG C; Reaction times is 0.5 ~ 24h, is preferably 2 ~ 8h;
Preferably, step c) described in positive charge modifier be amine or amine salt, be preferably NH
2r1, NHR1R2, NR1R2R3, R4-CO-NH-R5 or its customary salt, wherein R1, R2, R3, R4, R5 is selected from the alkane of substituted or unsubstituted C1 ~ 15 straight or branched respectively, substituted or unsubstituted C1 ~ 15 straight or branched alkene, the more preferably alkane of substituted or unsubstituted C2 ~ 12 straight or branched, substituted or unsubstituted C2 ~ 12 straight or branched alkene, be more preferably 2-diethylin monochloroethane hydrochloride, hexanediamine, diethylamine, trimethylammonium thanomin, N-[3-(dimethylamino) propyl group] Methacrylamide, polymine, Tri N-Propyl Amine, N-heptyl amice, N-amino dodecane or thanomin, described substituting group is selected from amino, hydroxyl or halogen, described positive charge modifier also comprises poly positive charge polymer, is preferably chitosan, polyetherimide or oligochitosan, crosslinking reaction temperature is 15 ~ 80 DEG C, cross-linking reaction time is 0.5 ~ 24h,
Preferably, step e) described in positively charged matrix microspheres and the mass ratio of protein/polypeptide be 2:1 ~ 100:1, be preferably 3:1 ~ 10:1; Described temperature of reaction is 15 ~ 80 DEG C, is preferably 35 ~ 50 DEG C; Reaction times is 0.5 ~ 24h, is preferably 2 ~ 8h; Mixing speed is 10 ~ 300rpm;
Preferably, step f) and step h) between can also comprise step g), step g) for step f) complete after system in add damping fluid and molecule crosslinked dose of protein/polypeptide, constant temperature stirring reaction, drain gained feed liquid after reaction terminates; Molecule crosslinked dose of described protein/polypeptide has the functional group can reacted with protein/polypeptide; The consumption that protein/polypeptide is molecule crosslinked dose is 0.1 ~ 100mmol/g microballoon, is preferably 1 ~ 50mmol/g microballoon; Described temperature of reaction is 15 ~ 80 DEG C, is preferably 35 ~ 50 DEG C; Reaction times is 0.5 ~ 24h, is preferably 2 ~ 8h; Mixing speed is 10 ~ 300rpm;
Preferably, at described step j) in, to step I) complete after gained matrix microspheres in can also add oxidation inhibitor, stirring reaction; The consumption of described linking agent is 0.1 ~ 100mmol/g microballoon, preferably 1 ~ 50mmol/g microballoon; Reaction times is 0.5 ~ 24h, is preferably 2 ~ 8h; Mixing speed is 10 ~ 300rpm.
9. the preparation method according to any one of claim 6 ~ 8, it is characterized in that, described method can also comprise being cross-linked between protein/polypeptide molecule, cross-linking reagent used is dialdehyde base class, bis-epoxy class, diimine class, nitrine quasi-molecule or the wherein mixture of at least two kinds, be preferably mda, suceinic aldehyde, carbodiimide, vulcabond, genipin, glutaraldehyde or nitrine diphenylphosphine or the wherein mixture of at least two kinds, more preferably suceinic aldehyde, carbodiimide, vulcabond or the wherein mixture of at least two kinds; The consumption of cross-linking reagent is 0.1 ~ 100mmol/g microballoon, is preferably 1 ~ 50mmol/g microballoon, more preferably 1 ~ 10mmol/g microballoon; Crosslinking reaction temperature is 15 ~ 65 DEG C; Cross-linking reaction time is 0.5 ~ 24h.
10. the application of the microcarrier according to any one of Claims 1 to 5 in cell cultures.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510090678.8A CN104694454B (en) | 2015-02-28 | 2015-02-28 | A kind of microcarrier for cell culture and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510090678.8A CN104694454B (en) | 2015-02-28 | 2015-02-28 | A kind of microcarrier for cell culture and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104694454A true CN104694454A (en) | 2015-06-10 |
| CN104694454B CN104694454B (en) | 2017-11-28 |
Family
ID=53342013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510090678.8A Active CN104694454B (en) | 2015-02-28 | 2015-02-28 | A kind of microcarrier for cell culture and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104694454B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108904890A (en) * | 2018-06-19 | 2018-11-30 | 北京科技大学 | The biomimetic porous microcarrier of Dynamic electrostatic deposition compounding natural material and preparation method |
| CN110177870A (en) * | 2017-01-20 | 2019-08-27 | 新加坡科技研究局 | Thermo-responsive micro-carrier system and application thereof |
| CN110484486A (en) * | 2019-08-05 | 2019-11-22 | 嘉兴千纯生物科技有限公司 | A kind of complex function type microcarrier and preparation method thereof |
| CN114058569A (en) * | 2021-11-19 | 2022-02-18 | 博格隆(浙江)生物技术有限公司 | Animal cell culture microcarrier and preparation method thereof |
| CN116262222A (en) * | 2023-01-09 | 2023-06-16 | 浙江纽龙合成材料制造有限公司 | Preparation method of diethylaminoethyl natural polysaccharide chromatographic medium |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5512474A (en) * | 1992-05-29 | 1996-04-30 | Bsi Corporation | Cell culture support containing a cell adhesion factor and a positively-charged molecule |
| CN101405384A (en) * | 2006-03-17 | 2009-04-08 | 三洋化成工业株式会社 | Cell culture substrate |
| CN101864391A (en) * | 2010-05-18 | 2010-10-20 | 博格隆(上海)生物技术有限公司 | Microcarrier for culturing animal cell and crosslinking method thereof |
| WO2012158108A1 (en) * | 2011-05-16 | 2012-11-22 | Ge Healthcare Bio-Sciences Ab | Method of cultivating cells on microcarriers in a bag |
-
2015
- 2015-02-28 CN CN201510090678.8A patent/CN104694454B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5512474A (en) * | 1992-05-29 | 1996-04-30 | Bsi Corporation | Cell culture support containing a cell adhesion factor and a positively-charged molecule |
| CN101405384A (en) * | 2006-03-17 | 2009-04-08 | 三洋化成工业株式会社 | Cell culture substrate |
| CN101864391A (en) * | 2010-05-18 | 2010-10-20 | 博格隆(上海)生物技术有限公司 | Microcarrier for culturing animal cell and crosslinking method thereof |
| WO2012158108A1 (en) * | 2011-05-16 | 2012-11-22 | Ge Healthcare Bio-Sciences Ab | Method of cultivating cells on microcarriers in a bag |
Non-Patent Citations (1)
| Title |
|---|
| BRUCE S.JACOBSON AND UNA S.RYAN: "GROWTH OF ENDOTHELIAL ANDHeLa CELLS ON A NEW MULTIPURPOSEMICROCARRIER THAT IS POSITIVE,NEGATIVE OR COLLAGEN COATED", 《TISSUE & CELL》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110177870A (en) * | 2017-01-20 | 2019-08-27 | 新加坡科技研究局 | Thermo-responsive micro-carrier system and application thereof |
| CN110177870B (en) * | 2017-01-20 | 2023-10-27 | 新加坡科技研究局 | Thermally responsive microcarrier system and use thereof |
| CN108904890A (en) * | 2018-06-19 | 2018-11-30 | 北京科技大学 | The biomimetic porous microcarrier of Dynamic electrostatic deposition compounding natural material and preparation method |
| CN110484486A (en) * | 2019-08-05 | 2019-11-22 | 嘉兴千纯生物科技有限公司 | A kind of complex function type microcarrier and preparation method thereof |
| CN114058569A (en) * | 2021-11-19 | 2022-02-18 | 博格隆(浙江)生物技术有限公司 | Animal cell culture microcarrier and preparation method thereof |
| CN116262222A (en) * | 2023-01-09 | 2023-06-16 | 浙江纽龙合成材料制造有限公司 | Preparation method of diethylaminoethyl natural polysaccharide chromatographic medium |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104694454B (en) | 2017-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104694454A (en) | Micro-carrier for cell culture as well as preparation method and application of micro-carrier | |
| CN102732475A (en) | Microcarrier used for cell culture, its preparation method and its detection method | |
| CN105492595B (en) | Cell culture article and method | |
| CN102068965B (en) | Method for preparing chitosan separation medium suitable for protein purification | |
| US4266032A (en) | Method of culturing microcarrier supported cells | |
| CN101405384A (en) | Cell culture substrate | |
| CN101892217A (en) | Preparation method of magnetic chitosan compound microsphere immobilized marine alkaline proteinase | |
| Van der Velden-deGroot | Microcarrier technology, present status and perspective | |
| CN103275923B (en) | A kind of GF microcarrier for cell culture and preparation method thereof | |
| CN101864391B (en) | Microcarrier for culturing animal cell and crosslinking method thereof | |
| CN106834204A (en) | A kind of cell culture SFL microcarriers and its preparation method and application | |
| EP0297605B1 (en) | Cell culture microcarriers | |
| CN103215217B (en) | A kind of animal cell culture collagen protein coating microcarrier and preparation method thereof | |
| US7670839B2 (en) | Process for coating cell-culture support | |
| EP2271739B1 (en) | Product for cell culture | |
| CN114058569B (en) | Animal cell culture microcarrier and preparation method thereof | |
| CN105154426B (en) | A kind of process for fixation of zytase | |
| CN102719480A (en) | Pre-processing method of cell culture plate for suspension cell liposome transfection | |
| CN114634961B (en) | Preparation method of oyster oligopeptide powder with multiple bioactivity functions | |
| Hamzah et al. | Synergistic effect of two type cellulase immobilized on chitosan microparticle as biocatalyst for coconut husk hydrolysis | |
| CN102703416B (en) | Preparation method of immobilized enzyme carrier | |
| CN1863819A (en) | Attachment of cells to surfaces | |
| JP7039308B2 (en) | Cell culture carrier | |
| AU2021106067A4 (en) | A Method for Modifying a Porcine Cholecyst Derived Scaffold by Coupling with Fibronectin for Skin Tissue Engineering Application | |
| CN104962543A (en) | Oriented immobilization method for bacillus subtilis neutral protease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |