CN103113627B - Polysaccharide/Matrigel compound gel membrane for cell culture as well as preparation method and application thereof - Google Patents
Polysaccharide/Matrigel compound gel membrane for cell culture as well as preparation method and application thereof Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a polysaccharide/Matrigel compound gel membrane for cell culture as well as a preparation method and an application thereof. The polysaccharide/Matrigel compound gel membrane contains polysaccharide and Matrigel and is prepared by tape casting based on calcium ions as a crosslinking agent. The method is simple in process, and the compound gel membrane prepared can simulate structure, composition, physical characteristics and functions of in vivo cell basement membrane so as to facilitate proliferation of various cells. Different crosslinking agents in different use levels can be selected according to types of cells to adjust the rigidity of the basement so as to adjust the adhering and differentiating behaviors of cells. Meanwhile, the compound gel membrane has good mechanical properties and is suitable for long-term culture of cells. The compound gel membrane is relatively low in cost and safer to apply.
Description
Technical field
The present invention relates to the technical field of cell cultures, be specifically related to a kind of polysaccharide for cell cultures/Matrigel plural gel film and its preparation method and application.
Background technology
Cell amplification is the prerequisite of cell therapy and organizational project development; the New Measure of the multiple pilot scale culture attached cell of development in recent years; widespread use in the large scale culturing such as engineering cell system; namely by analog cell growth microenvironment in vivo and propagation activity, the basic point of departure that its amplification and directed differentiation are culturing cells is controlled.Multiplex at present have good biocompatibility material and carry out analog cell epimatrix, promotes the amplification in vitro of cell, mainly comprise hyaluronic acid, collagen, chitosan, alginates, gelatin etc.In recent years, Matrigel receives increasing concern due to the performance of its uniqueness in cell cultures, Matrigel isolates in EHS mouse tumor, mainly comprise the compositions such as ln, type Ⅳ collagen, nidogen, heparin sulfate glycoprotein, also include the somatomedin of some trace, Urogastron, transforming growth factor-beta, fibroblast growth factor etc.Be applicable at the bottom of the substratum as various kinds of cell, build Various Tissues.But it is expensive, simultaneously its inconvenient operation, makes its large-scale application on cell amplification is cultivated receive certain restriction.
Summary of the invention
For solving the problem, the invention provides a kind of polysaccharide for cell cultures/Matrigel plural gel film and preparation method thereof, plural gel film obtained in this way contains the polysaccharide and Matrigel with biocompatibility, can the structure of analogue body inner cell basilar membrane, composition, physical property and function, the propagation of various kinds of cell can be promoted.
The present invention is realized by following technical proposals:
A kind of polysaccharide for cell cultures of the present invention/Matrigel plural gel film, is characterized in that with polysaccharide and Matrigel for raw material, adopts calcium ion as linking agent, by Liu Yanfa (also claiming " casting method ") preparation.
In the present invention, preferably, described polysaccharide is the combination of one or more polysaccharide in alginates, pectin, hyaluronic acid, chondroitin sulfate.
In the present invention, preferably, described polysaccharide/Matrigel plural gel film prepares by the following method:
(1) take a certain amount of polysaccharide, be dissolved in deionized water, being prepared into mass percent concentration is 0.1%-3% polysaccharide soln, stirs until all dissolve, for subsequent use;
(2) calcium chloride solution of 0.1M is prepared, filtration sterilization, for subsequent use;
(3) (adding a certain amount of deionized water wherein, to form protein concentration be the Matrigel solution of 9-15mg/ml, and whole operating process need operate on ice for self-control or purchased from BD company, article No.: 354234) to get the Matrigel solution of certain volume;
(4) in Bechtop, measure solution 5ml prepared by step (1), add Matrigel solution 0.1-8ml prepared by step (3) wherein, add the calcium chloride solution that 0.5-5ml step (2) is prepared, mix and blend, then be poured in cell culture vessel or Tissue Culture Plate, above process need operate on ice, is then placed in 37 DEG C of incubators and hatches 2-5min, forms the plural gel film being used for cell cultures.
The protein concentration (gum concentration) of 9mg/ml be Matrigel at a certain temperature (22-35 DEG C) oneself form the minimum concentration of gel, form gel in the present invention by two kinds of modes:
(1) rely on polysaccharide and linking agent (calcium chloride) chemical reaction occurs and is formed, when in composite membrane, matrigel comparision contents is low, mainly through being cross-linked to form gel with polysaccharide, Matrigel can form half interpenetrating network structure with polysaccharide and form plural gel;
(2) then dual network gel is jointly formed with polysaccharide when Matrigel concentration reaches 9mg/ml or more.
Further, the invention allows for a kind of method preparing the described polysaccharide for cell cultures/Matrigel plural gel film, it is characterized in that comprising the following steps:
(1) take a certain amount of polysaccharide, be dissolved in deionized water, being prepared into mass percent concentration is 0.1%-3% polysaccharide soln, stirs until all dissolve, for subsequent use;
(2) calcium chloride solution of 0.1M is prepared, filtration sterilization, for subsequent use;
(3) (adding a certain amount of deionized water wherein, to form protein concentration be the Matrigel solution of 9-15mg/ml, and whole operating process need operate on ice for self-control or purchased from BD company, article No.: 354234) to get the Matrigel solution of certain volume;
(4) in Bechtop, measure solution 5ml prepared by step (1), add Matrigel solution 0.1-8ml prepared by step (3) wherein, add the calcium chloride solution that 0.5-5ml step (2) is prepared, mix and blend, then be poured in cell culture vessel or Tissue Culture Plate, above process need operate on ice, is then placed in 37 DEG C of incubators and hatches 2-5min, forms the plural gel film being used for cell cultures.
In the present invention, preferably, described polysaccharide is the combination of one or more polysaccharide in alginates, pectin, hyaluronic acid, chondroitin sulfate.
Simple and the plural gel film prepared by the method for the method process, the propagation of various kinds of cell can be promoted, this plural gel film has good mechanical property simultaneously, the long-term cultivation of cell can be applicable to, and different Matrigel content can be selected to regulate and control the composition of basilar membrane according to cell type, select the consumption of different linking agent to regulate substrate rigidity with the adhesion of regulating cell and differentiation behavior.
Therefore, further, the present invention proposes the described polysaccharide for cell cultures/Matrigel plural gel film to apply in culturing cell.
Accompanying drawing explanation
Fig. 1 is the figure of cardiac stem cells when sodium alginate/Matrigel plural gel film surface is cultivated 3 days;
Fig. 2 is the figure of cardiac stem cells when hyaluronic acid/Matrigel plural gel film surface is cultivated 3 days.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation of embodiment 1 sodium alginate/Matrigel plural gel film
(1) take 0.1g sodium alginate in 9.9ml deionized water, being prepared into mass percentage concentration is 1% sodium alginate soln, stirs until all dissolve, for future use;
(2) prepare the calcium chloride solution of 0.1M, filtration sterilization is for subsequent use;
(3) get the self-control Matrigel solution (preparing according to the method for embodiment 5) of certain volume, add a certain amount of deionized water wherein and form the Matrigel solution that protein concentration is 9mg/ml, whole operating process need operate on ice;
(4) in Bechtop, measure solution 5ml prepared by step (1), add Matrigel solution 4ml prepared by step (3) wherein, add the calcium chloride solution that 1ml step (2) is prepared, mix and blend, then be poured in cell culture vessel or Tissue Culture Plate, above process need operate on ice, is then placed in 37 DEG C of incubators and hatches 2-5min, just can form the plural gel film for cell cultures.
The preparation of embodiment 2 hyaluronic acids/Matrigel plural gel film
(1) take 0.3g hyaluronic acid in 9.7ml deionized water, being prepared into mass percentage concentration is 3% hyaluronic acid solution, stirs until all dissolve, for future use;
(2) prepare the calcium chloride solution of 0.1M, filtration sterilization is for subsequent use;
(3) get certain volume Matrigel solution (purchased from BD company, article No.: 354234), adding a certain amount of deionized water wherein, to form protein concentration be the Matrigel solution of 15mg/ml, and whole operating process need operate on ice;
(4) in Bechtop, measure solution 5ml prepared by step (1), add Matrigel solution 4ml prepared by step (3) wherein, add the calcium chloride solution that 0.5ml step (2) is prepared, mix and blend, then be poured in cell culture vessel or Tissue Culture Plate, above process need operate on ice, is then placed in 37 DEG C of incubators and hatches 2-5min, just can form the plural gel film for cell cultures.
The preparation of embodiment 3 chondroitin sulfates/Matrigel plural gel film
(1) take 0.2g chondroitin sulfate in 9.8ml deionized water, being prepared into mass percentage concentration is 2% chondroitin sulfate solution, stirs until all dissolve, for future use;
(2) prepare the calcium chloride solution of 0.1M, filtration sterilization is for subsequent use;
(3) get the self-control Matrigel solution (preparing according to the method for embodiment 5) of certain volume, add a certain amount of deionized water wherein and form the Matrigel solution that protein concentration is 9mg/ml, whole operating process need operate on ice;
(4) in Bechtop, measure solution 5ml prepared by step (1), add Matrigel solution 2ml prepared by step (3) wherein, add the calcium chloride solution that 2ml step (2) is prepared, mix and blend, then be poured in cell culture vessel or Tissue Culture Plate, above process need operate on ice, is then placed in 37 DEG C of incubators and hatches 2-5min, just can form the plural gel film for cell cultures.
The preparation of embodiment 4 pectin/Matrigel plural gel film
(1) take 0.2g pectin in 9.8ml deionized water, being prepared into mass percentage concentration is 2% pectin solution, stirs until all dissolve, for future use;
(2) prepare the calcium chloride solution of 0.1M, filtration sterilization is for subsequent use;
(3) get the self-control Matrigel solution (preparing according to the method for embodiment 5) of certain volume, add a certain amount of deionized water wherein and form the Matrigel solution that protein concentration is 9mg/ml, whole operating process need operate on ice;
(4) in Bechtop, measure pectin solution 5ml prepared by step (1), add Matrigel solution 2ml prepared by step (3) wherein, add the calcium chloride solution that 2ml step (2) is prepared, mix and blend, then be poured in cell culture vessel or Tissue Culture Plate, above process need operate on ice, is then placed in 37 DEG C of incubators and hatches 2-5min, just can form the plural gel film for cell cultures.
The preparation of embodiment 5Matrigel
One, for a pipe EHS cell sample (1.5ml)
1, material prepares: female C57BL/6 mouse, PBS, 1ml(16G syringe needle)
2, step:
1) on ice or 4 DEG C of refrigerators melt (selecting 4 DEG C of refrigerators, temperature-controllable)
2) anesthetized mice: (carrying out in stink cupboard), by being soaked with the paper handkerchief of saturated ether as bottom large beaker, puts into mouse.Ether is not directly spread across on mouse.
3) every injected in mice 0.5ml EHS cell is in right back outer side.Needle distal end is inserted main muscle masses, and sarcoma should be injected in hindlimb muscle.
4) mark cage, make a record, cleaning experiment appliance and laboratory.
5) carry out EHS after 3-4 week to go down to posterity.
Two, EHS sarcoma goes down to posterity and Collection and conservation
(1) material prepares:
1. dissect apparatus-little scissors, tweezers (in advance autoclaving)
2.6ml/20-30ml syringe: 16G syringe needle
3.50ml centrifuge tube, beaker, ice bucket is multiple, dry ice
4.PBS, 70% ethanol, ether, 4 DEG C of whizzers, the small beaker that sterilizing is good
5. thieving paper, institute's pocket-preservation sarcoma and process mouse are used
(2) the sarcoma preparation of going down to posterity
Precaution
1. get out a large amount of ice and the PBS of precooling, during operation, sample is on ice as far as possible
2.EHS must bacteriological infection be noted in the process gone down to posterity of tumour
1) abandon after there is region that is watery, that turn to be yellow and extract in tumor region
2) antibiotic utilization
3) each tumour separate operation in succeeding generations
Step:
1. dry ice puts to death mouse: add dry ice in small beaker, put into large beaker: add a few ml water to dry ice, mouse is put into large beaker.
Get out 70% ethanol in 2.1L large beaker, upper cover preservative film, the mouse after execution is immersed, and carries out disinfection and can prevent mouse hair-videotape in experiment below.
3. carefully around sarcoma, break skin, raise Dermal exposure sarcoma.
4. sarcoma is carefully taken off by little scissors and tweezers, puts into 50ml centrifuge tube, and every murine sarcoma is deposited separately, and centrifuge tube is numbered.
The PBS of 20-25ml precooling is added, concussion in 5 each pipes.
6 mixtures step 5 obtained are poured in 20-30ml syringe, disperse sarcoma cell subsequently
1) miscellany is pasted centrifuge tube inwall to push back in 50ml centrifuge tube
2) pour mixture into syringe again, and load onto 16G syringe needle.
3) push mixture and return 50ml centrifuge tube.
Necessary words can operate once again
7 to add PBS to total amount be 40ml, fully shakes dispersion tissue.
Centrifuge tube is put into whizzer by 8, opens whizzer, when rotating speed disconnects immediately to during 1000rpm
9 outwell supernatant, add 40ml PBS, fully jolt.
10 repeating steps 8
11 every 10ml mixtures add about 1ml mycillin and mix.Remain before injection that sample is on ice.5%DMSO can be added frozen.
(3) sarcoma is collected
Precaution
1. do not allow the too large of tumor growth, otherwise easily necrose, Matrigel production declining after tissue necrosis.
2. the tumour containing a large amount of blood and yellow extract can not be used.
3. the tumour generally, being greater than 4 grams is easily downright bad
Step:
1. dry ice is put to death in mouse-small beaker and is added dry ice, puts into large beaker; Add a few ml water to dry ice, mouse is put into large beaker
2., after dead mouse, they are soaked in 70% ethanol.
3. cut off skin, expose sarcoma.
4. sarcoma closed in large beaker to remove and touched, beaker is as on ice.
5., after all sarcomas are all collected, by it mixing, make Matrigel immediately
Make M:100g sarcoma amount
(1) material prepares:
1.50ml centrifuge tube, the large beaker of dialysis: stirrer: dialysis tubing (pre-treatment), a large amount of ice
2. solution: 3.4M NaCl solution; 2M urea damping fluid; Tris-saline; Chloroform; 1% high sugar soln
3. mosquito forceps and scissors shift to an earlier date autoclaving
4. preserve the sterile chamber of Matrigel
(2) precaution
1. all reagent, articles for use and appliance requires 4 DEG C of precoolings.In whole preparation process, organize as on ice, subzero treatment.
Should be quick as much as possible in 2.Matrigel preparation process, otherwise its active reduction, whole separation and dialysis will complete in 4 days.
3. once isolate Matrigel, 4 DEG C of preservations, because it is once solidify, will not in liquefaction
The final protein concentration of 4.Matrigel is not less than 9mg/ml(9-15mg/ml)
When 5.Matrigel concentration is too low, the method for ammonium sulfate precipitation can be used to concentrate, then dialyse.
(3) step:
1.(washs) add 3.4M NaCl solution 200ml in sarcoma, homogenate is until dispersion.
Homogenization time can not be oversize, in order to avoid temperature is too high make proteolytic degradation.
2.7000rpm4 DEG C of centrifugal 15min, abandons pink supernatant.
3. repeating step 1,2 twice.
4. add 2M urea damping fluid 100ml in sarcoma to mix
5.4 DEG C to stir and spend the night.
6.14000rpm4 DEG C of centrifugal 20min, retains supernatant on ice.
7. adding 2M urea damping fluid 50ml in sarcoma resistates mixes.
8.14000rpm4 DEG C of centrifugal 20min, retains supernatant on ice.
9. the supernatant that obtains of mixing step 6 and step 8, use 2L Tris dialysis, 1L adds 5ml chloroform, sterilization
10. dialyse more than 4 hours, rotate sack at end
11. add separately Tris does not add chloroform process more than 2 hours
12. repeating steps 11
Salt medium is cancelled in 13. dialysis for the last time, is changed to: 1%(w/w) high sugar soln
14. in super clean bench, and mosquito forceps fixes dialysis tubing, after 70% alcohol flushing, is cut off by sack with scissors, is transferred to by extract in sterile chamber, must be monoblock, operates in and carries out on ice.
15. packing immediately, cooling, preserve 1 year for-20 DEG C
Gelling action: be poured over by extract in required container, rewarming 30-120min spreads thin layer.
Application Example 1
By cardiac stem cells with 1 × 10
4individual/cm
2density is planted in sodium alginate/Matrigel composite film surface that embodiment 1 prepares respectively and hyaluronic acid/Matrigel composite film surface that embodiment 2 prepares is cultivated 3 days, changed liquid every other day.When cell is paved with whole gel-film, be placed on 4 DEG C, 15min, add a small amount of pancreatin, centrifugal collecting cell.
Meanwhile, according to method identical above by cardiac stem cells with 1 × 10
4individual/cm
2density to be planted on ordinary cells growth plate as a control group.Basis of microscopic observation cell growth status, Fig. 1 is cardiac stem cells figure when growing 3 days on the sodium alginate that embodiment 1 prepares/Matrigel composite film surface; Fig. 2 is cardiac stem cells figure when growing 3 days on the hyaluronic acid that embodiment 2 prepares/Matrigel composite film surface.Can find out that the cardiac stem cells when polysaccharide of the present invention/composite membrane upper surface grows 3 days shows good growth conditions from microscopy results, and there is the specific function of cardiac stem cells, and along with the increase of Matrigel content, the adhesive capacity of cardiac stem cells is better.The cardiac stem cells that ordinary cells culture plate grows then occurs spherical and growth is slower.
Claims (5)
1. for polysaccharide/Matrigel plural gel film of cell cultures, it is characterized in that with polysaccharide and Matrigel for raw material, adopt calcium ion as linking agent, prepared by Liu Yanfa:
(1) take a certain amount of polysaccharide, be dissolved in deionized water, being prepared into mass percent concentration is 0.1%-3% polysaccharide soln, stirs until all dissolve, for subsequent use;
(2) calcium chloride solution of 0.1M is prepared, filtration sterilization, for subsequent use;
(3) get the Matrigel solution of certain volume, add a certain amount of deionized water wherein and form the Matrigel solution that protein concentration is 9-15mg/mL, whole operating process need operate on ice;
(4) in Bechtop, measure solution 5mL prepared by step (1), add Matrigel solution 0.1-8mL prepared by step (3) wherein, add the calcium chloride solution that 0.5-5mL step (2) is prepared, mix and blend, then be poured in cell culture vessel or Tissue Culture Plate, above process need operate on ice, is then placed in 37 DEG C of incubators and hatches 2-5min, forms the plural gel film being used for cell cultures.
2. plural gel film as claimed in claim 1, is characterized in that described polysaccharide is the combination of one or more polysaccharide in alginates, pectin, hyaluronic acid, chondroitin sulfate.
3. the method for the polysaccharide/Matrigel plural gel film of preparation described in claim 1 or 2, is characterized in that comprising the following steps:
(1) take a certain amount of polysaccharide, be dissolved in deionized water, being prepared into mass percent concentration is 0.1%-3% polysaccharide soln, stirs until all dissolve, for subsequent use;
(2) calcium chloride solution of 0.1M is prepared, filtration sterilization, for subsequent use;
(3) get the Matrigel solution of certain volume, add a certain amount of deionized water wherein and form the Matrigel solution that protein concentration is 9-15mg/mL, whole operating process need operate on ice;
(4) in Bechtop, measure solution 5mL prepared by step (1), add Matrigel solution 0.1-8mL prepared by step (3) wherein, add the calcium chloride solution that 0.5-5mL step (2) is prepared, mix and blend, then be poured in cell culture vessel or Tissue Culture Plate, above process need operate on ice, is then placed in 37 DEG C of incubators and hatches 2-5min, forms the plural gel film being used for cell cultures.
4. prepare the method for plural gel film as claimed in claim 3, the polysaccharide that it is characterized in that described in step (1) is the combination of one or more polysaccharide in alginates, pectin, hyaluronic acid, chondroitin sulfate.
5. the polysaccharide described in claim 1 or 2/Matrigel plural gel film is applied in culturing cell.
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| CN101679948A (en) * | 2007-04-20 | 2010-03-24 | 弗劳恩霍芬-格泽尔沙夫特祖尔弗德伦德尔安格万特福施有限公司 | Cell culture system |
| CN101855340A (en) * | 2007-03-06 | 2010-10-06 | 北卡罗来纳大学查珀尔希尔分校 | Complex of hyaluronic acid, other matrix components, hormones and growth factors for cell maintenance, expansion and/or differentiation |
| CN102337259A (en) * | 2011-10-24 | 2012-02-01 | 上海交通大学医学院附属瑞金医院 | A kind of microencapsulated human pancreatic cancer tumor cell and its preparation method and application |
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| CN101679948A (en) * | 2007-04-20 | 2010-03-24 | 弗劳恩霍芬-格泽尔沙夫特祖尔弗德伦德尔安格万特福施有限公司 | Cell culture system |
| CN102337259A (en) * | 2011-10-24 | 2012-02-01 | 上海交通大学医学院附属瑞金医院 | A kind of microencapsulated human pancreatic cancer tumor cell and its preparation method and application |
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