CN1392253A - Method of utilizing three-dimensional rack seed cell double phase inoculation to construct engineered tissue - Google Patents
Method of utilizing three-dimensional rack seed cell double phase inoculation to construct engineered tissue Download PDFInfo
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- CN1392253A CN1392253A CN02133401A CN02133401A CN1392253A CN 1392253 A CN1392253 A CN 1392253A CN 02133401 A CN02133401 A CN 02133401A CN 02133401 A CN02133401 A CN 02133401A CN 1392253 A CN1392253 A CN 1392253A
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Abstract
The present invention belongs to biomedicine engineering and is one method of constructing engineered tissue. The present invention features that biogel liquid is first prepared and them mixed with seed cell to form cell-biogel suspension, which exists first in liquid state and then in solid in certain condition, to realize double phase inoculation of seed cell in 3D rack. The tree parts of seed cell, biogel and 3D rack forms one united integral and this can realize the efficient combination between cell and material without loss or dropping, maintain the 3D homogeneous distribution of cell in material and maintain the initial form of the engineered tissue. The method of the present invention has simple operation, easy control and short culture period.
Description
One, technical field
The invention belongs to biomedical engineering, be specifically related to make up the method for engineering tissue.
Two, background technology
The Tissue Engineering Study of multiple histoorgans such as human body cartilage, bone, cornea, skin, muscle and liver is the problem that receives much attention in the current biomedical engineering, and utilization biomaterial technique construction engineering tissue is the core content of Tissue Engineering Study.In the research that makes up engineering tissue, find, because three-dimensional porous crack timbering material is to the consistency deficiency of seed cell, material only provides limited reasons such as surface-area for seed cell adheres to, exist and lose after the seed cell inoculation and come off in a large number, cell and three-dimensional rack composite efficiency hang down inferior problem; In addition, cell is still two-dimensional growth in timbering material, is unfavorable for cellular-restoring or keeps its phenotype and synthetic and secretory cell epimatrix.Combine its above-mentioned result, make the structure effect of engineering tissue not good enough exactly, vitro culture is difficult to generate the engineering tissue with typical organization's structure.For solving the problem that seed cell comes off, the foreign study person has carried out multiple different exploration.For example, aspect the structure of cartilage engineering tissue, document 1:FreedLE is arranged, HollanderAP, Matin I, et al.Chondrogenesis in a cell-pilymer-bioreactorsystem.Exp Cell Res, 1998,240:58-65; With document 2:Ma PX, LangerR.Morphology and mechanical function or Long-term in vitro engineeredcartilage.J Biomed Mater Re, 1999,44:217-221. its technical characterictic is to be 1cm with diameter, high 0.2cm, porosity is that to be dipped in cell density be 5 * 10 to 95% timbering material
5In chondrocyte's suspension of individual/ml, magnetic stirring bar with long 4cm stirs with the speed that per minute 50-60 changes, in Erlenmeyer flask, cultivate after 3-6 days under 37 ℃, the condition of 5%CO2 and be transferred in the rotary microgravity culture systems of 110ml, cultivate and 8 weeks formed cartilage with certain weave construction, the weave construction of periphery is better than central part, cultivates 12 weeks can produce more sophisticated cartilaginous tissue later.There is following defective in above-mentioned prior art: complicated operation, need special-purpose bio-reactor, and the recombination rate of cell and timbering material is low, and cell still is two-dimensional growth in support, and vitro culture generates the time long (8-12 week) that more sophisticated tissue needs.Also has document 3:Brun P, Abatangelo G, Radice M, et al.Chondrocyteaggregation and reorganization into three-dimensional scaffolds.JBiomed Mater Res, 1999,46:337-346; With document 4:Aigner J, Tegeler J, HutzlerTP, et al.Cartilage tissue engineering with novel nonwoven structuredbiomaterial based on hyaluronic acid benzyl ester.J Biomed MaterRes, 1998, its technical characterictic of 42:172-181. be respectively with fusion method with 2-3 * 10
4Individual cell and 4 * 10
6Individual cell inoculation is in diameter 10mm, thick 2mm hyaluronic acid fibrous framework, hatched 3 hours and 5 hours, cell has good adhesion to support, but cell is in support and is dispersed in heterogeneous distribution, in the culture dish of 21 days and 33 days, cultivate, whole cell one supporting structure can not form the cartilaginous tissue structure of homogeneous, and the cell mass that only has some to assemble growth in the support can form cartilage structure.It still needs special-purpose bio-reactor, and the recombination rate of cell and timbering material is low, and cell still is two-dimensional growth in support, and vitro culture can not generate the complete tissue of the three-dimensional structure consistent with timbering material.
Three, summary of the invention
The objective of the invention is to defective at above-mentioned prior art, adopt the method for three-dimensional rack seed cell double phase inoculation to make up engineering tissue, make it easy and simple to handle, controllability is good, cell and support can be efficiently compound, cell is uniform three dimensional and distributes, makes up that little block organization need not bio-reactor, to obtain the required incubation time of mature tissue short, tissue three-dimensional profile that is generated and timbering material basically identical in support.
Technical scheme of the present invention is as follows:
A kind of method of utilizing three-dimensional rack seed cell double phase inoculation to construct engineered tissue is made up of the cultivation six step process processes of modulation, cell-xanthan gel suspension and the inoculation of organizational project three-dimensional rack and the tissue block of the cultivation amplification of the sterilization aquation of the preparation of xanthan gel liquid, organizational project three-dimensional stent material, cell and collection, cell-xanthan gel suspension.
1. the preparation of xanthan gel liquid
Under aseptic condition, cut tendon tissue and with its pulverizing, be soaked in the Glacial acetic acid of 0.010-0.001 volume ratio 72 hours, fully vibrated once in every 6-8 hour, centrifugal with the speed that per minute 10000-15000 changes, get supernatant liquor, there is precipitation to separate out when adding 0.1N NaOH to PH=6.5-7.5, centrifugal with the speed of per minute 10000-15000 commentaries on classics again, abandon supernatant liquor, the Glacial acetic acid that adds an amount of 0.01-0.001 volume ratio dissolves throw out, make the liquid xanthan gel of preliminary purification, the sample that takes a morsel is measured protein content, and all the other xanthan gel liquid packing are sealed up for safekeeping, mark time protein concentration is preserved down at-20 ℃.
2. the sterilization aquation of organizational project three-dimensional stent material
To select the aperture for use be 250 μ m-450 μ m, porosity greater than 85% tissue engineering bracket material, and material is sealed up for safekeeping with the oxirane disinfection film; If hydrophobic material with 75% alcohol immersion 10-40 minute, is used rinsed with deionized water 3 times again, cell culture fluid rinsing 3 times is drawn in the storage bottle that the nutrient solution that is full of in the timbering material is placed on lid with suction pipe and to be preserved temporarily.
3. the cultivation of cell amplification and collection
Seed cell is cultivated requirement condition by it to be inoculated in the culturing bottle, putting into CO2gas incubator cultivates, when treating that the cell growth reaches 90% fusion, with mass concentration is that 0.25% trypsinase/0.02%EDTA-Na2 digests collecting cell, with blood cell counting plate counting harvested cell sum, calculate viable cell per-cent with trypanblue exclusion method, calculate the viable cell sum again.
4. the modulation of cell one biological gel suspension
Get an amount of xanthan gel liquid, it is diluted to the desired protein concn of determining by the requirement of constructed tissue with the Glacial acetic acid of 0.01-0.001 volume ratio; Under ice bath, make gel alkalization regulator solution with 0.1NNaOH and 0.01 NaOH solution, the pH value of xanthan gel liquid is adjusted to 7.2-7.5; The nutrient solution and the serum of this neutrophilous organism coagulant liquid, 10 times of concentration are mixed in 8: 1: 1 ratios, obtain the pre-state xanthan gel that coagulates; Volume according to constructed tissue, ask for the pre-xanthan gel and the viable cell of coagulating of aequum respectively, dress viable cell and not having in the 50mL centrifuge tube of nutrient solution in gel joined fully is mixed with dropper piping and druming under the ice bath, obtains cell-xanthan gel suspension at last.
5. cell-xanthan gel suspension and organizational project three-dimensional rack inoculation
The organizational project three-dimensional stent material of sterilization or aquation is placed culture dish, cell-xanthan gel suspension is dripped in the organizational project three-dimensional stent material again, the cell one biological gel suspension to material reaches capacity; The culture dish that forms cell one gel one support complex body was inserted in 37 ℃, 5%CO2 incubator 10-15 minute; Add the desired nutrient solution of tissue culture, and break away from the bottom of making integration engineering tissue block and culture dish, all change nutrient solution after 3-5 hour and continue to cultivate.
6. the cultivation of tissue block
Tissue block is inserted in the culture dish, added desired nutrient solution, again culture dish is inserted in 37 ℃, 5%CO2 incubator and cultivate, changed nutrient solution once in per two days, to obtaining sophisticated tissue block.
The inventive method makes full use of biology (collagen) gel and exists with liquid form earlier, solidified characteristics under certain condition subsequently, realize three-dimensional rack seed cell double phase inoculation, make seed cell, xanthan gel, the three-dimensional stent material three forms unified integral body, so just solved the technical barrier that seed cell is lost and come off in the engineering tissue building process fully, thereby realized efficiently compound (100%) of cell and material well, keep the three-dimensional homogeneous phase of cell in material and distributed, and kept making up the original shape of engineering tissue; The inventive method is easy and simple to handle, and controllability is good, makes up little block organization (diameter is less than 1.5cm, and higher primary school is in 0.3cm) and need not bio-reactor; It is short to form the needed time of mature tissue, and the inventive method is applied to the external structure of cartilage engineering tissue, cultivates typical organization's structure that can generate cartilage 2 weeks under general conventional culture condition.
Four, description of drawings
Fig. 1 is cell-gel of the present invention-support composite curing photo substantially
Fig. 2 is a photo (* 100) under cell-gel of the present invention-support composite curing inverted microscope
Fig. 3 is the cardinal principle photo that cell-gel of the present invention-support complex body cultivated for two weeks
Fig. 4 is that cell-gel of the present invention-support complex body is cultivated two all histology pictures (toluidine blue dyeing * 100)
Five, embodiment
The inventive method is applied to the external structure of cartilage engineering tissue, the specific embodiment of the present invention is described.
1. the preparation of xanthan gel liquid:
Under aseptic condition, cut the tendon fibrous tissue of big white mouse tail, and will be cut into 1-2mm, be soaked in the Glacial acetic acid of 0.001 volume ratio 72 hours, fully vibration was once in per 8 hours.The speed of 10000-15000 commentaries on classics/per minute kind is centrifugal, get supernatant, adding when 0.1N sodium hydroxide is adjusted to 6.5-7.5 with pH value has precipitation to separate out, centrifugal with the speed of 10000-15000 commentaries on classics/per minute again, abandon supernatant, add the Glacial acetic acid of an amount of 0.001 volume ratio, with resolution of precipitate, make the liquid xanthan gel of preliminary purification, the sample that takes a morsel is measured protein content, and all the other xanthan gel packing are sealed up for safekeeping, mark time protein concentration ,-20 ℃ of preservations.
2. the sterilization aquation of organizational project three-dimensional stent material
Adopt calcium polyphosphate fiber (calium polyphosphate fiber, CPPf) and poly(lactic acid) (poly-L-lactic acid, PLLA) compound support frame material, two kinds of material volumes ratio: CPPf is 70%, PLLA is 30%, porosity is greater than 92%, the aperture is 250um-450um, seals up for safekeeping with the oxirane disinfection film; Other hydrophobic material the alcohol immersion 20-30 with 75% minute, with rinsed with deionized water three times, cell culture fluid rinsing three times, is drawn the storage bottle that the nutrient solution that is full of in the timbering material places lid with suction pipe.
3. the cultivation of cell amplification and collection
Articular chondrocytes is inoculated in 30mL with 3 * 104/cm2 contains (NUNCLON company in DMEM/Ham ' the s-F12 nutrient solution 175cm2 culturing bottle of " comprising that mass concentration is 10% foetal calf serum (Hyclone), 584mg/1L-glutamine, 0.1mM non-essential amino acid (Gibico), 0.4mM proline(Pro), 50ug/l vitamins C (Sigma) ", Germany), placing 37 ℃, volume fraction is in the 5%CO2 incubator; When cell grows to about 90% when merging, with mass concentration is that the chondrocyte is collected in 0.25% trypsinase/0.02%EDTA-Na2 digestion, with blood cell counting plate counting harvested cell sum, calculate viable cell per-cent with trypanblue exclusion method, and then calculate the viable cell sum.
4. the modulation of cell-xanthan gel suspension
Get an amount of xanthan gel liquid, being diluted to desired protein concn with the Glacial acetic acid of 0.001 volume ratio is 0.1%; Under ice bath, the pH value of regulator solution with xanthan gel liquid is adjusted to 7.2-7.5 with alkalizing, the DMEM nutrient solution and the serum of this neutrophilous organism coagulant liquid, 10 times of concentration are mixed in 8: 1: 1 ratios, obtain the pre-state xanthan gel that coagulates, it can keep under ice bath liquid 30 minutes; Get the pre-xanthan gel and the viable cell of coagulating of aequum respectively, dress viable cell and not having in the 50mL centrifuge tube of nutrient solution in gel is joined fully is mixed with dropper piping and druming under the ice bath, obtains chondrocyte-biology (collagen) gel suspension at last.
5. chondrocyte-collagen gel suspension and organizational project three-dimensional rack inoculation
The organizational project three-dimensional stent material of sterilization aquation is placed culture dish, cell-collagen gel suspension is dripped in the organizational project three-dimensional rack again, the cell-collagen gel suspension to material reaches capacity; The culture dish that will form cell-gel-support complex body placed 37 ℃, 5%CO2 incubator 10-15 minute, add the desired nutrient solution of tissue culture, and break away from the bottom of making integration engineering tissue block and culture dish, all change nutrient solution after 5 hours and continue to cultivate.
6. the cultivation of tissue block
Place culture dish to add desired an amount of nutrient solution tissue block, culture dish is inserted in 37 ℃, 5%CO2 incubator again and cultivate, per two change liquid greatly once, cultivate 2 weeks to obtain mature tissue's pieces.
Claims (7)
1, a kind of method of utilizing three-dimensional rack seed cell double phase inoculation to construct engineered tissue is characterized in that it is made up of the cultivation six step process processes of modulation, cell one biological gel suspension and the inoculation of organizational project three-dimensional rack and the tissue block of the cultivation amplification of the sterilization aquation of the preparation of xanthan gel liquid, organizational project three-dimensional stent material, cell and collection, cell one biological gel suspension.
2, make up the method for engineering tissue according to claim 1, the preparation that it is characterized in that said xanthan gel liquid is under aseptic condition, cut tendon tissue and with its pulverizing, be soaked in the Glacial acetic acid of 0.010-0.001 volume ratio 72 hours, fully vibrated once in every 6-8 hour, centrifugal with the speed that per minute 10000-15000 changes, get supernatant liquor, there is precipitation to separate out when adding 0.1NNaOH to PH=6.5-7.5, centrifugal with the speed of per minute 10000-15000 commentaries on classics again, abandon supernatant liquor, the Glacial acetic acid that adds an amount of 0.01-0.001 volume ratio dissolves throw out, makes the liquid xanthan gel of preliminary purification, and the sample that takes a morsel is measured protein content, all the other xanthan gel liquid packing are sealed up for safekeeping, mark time protein concentration is preserved down at 20 ℃.
3, make up the method for engineering tissue according to claim 1, the sterilization aquation that it is characterized in that said organizational project three-dimensional stent material is that to select the aperture for use be 250 μ m-450 μ m, porosity greater than 85% tissue engineering bracket material, and material is sealed up for safekeeping with the oxirane disinfection film; If hydrophobic material with 75% alcohol immersion 10-40 minute, is used rinsed with deionized water 3 times again, cell culture fluid rinsing 3 times is drawn in the storage bottle that the nutrient solution that is full of in the timbering material is placed on lid with suction pipe and to be preserved temporarily.
4, make up the method for engineering tissue according to claim 1, the cultivation amplification and the collection that it is characterized in that said cell are cell to be cultivated requirement condition by it be inoculated in the culturing bottle, putting into CO2gas incubator cultivates, when treating that the cell growth reaches 90% fusion, with mass concentration is that 0.25% trypsinase/0.02%EDTA-Na2 digests collecting cell, with blood cell counting plate counting harvested cell sum, calculate viable cell per-cent with trypanblue exclusion method, calculate the viable cell sum again.
5, make up the method for engineering tissue according to claim 1, the modulation that it is characterized in that said cell one biological gel suspension is to get an amount of xanthan gel liquid, with the Glacial acetic acid of 0.01-0.001 volume ratio it is diluted to the desired protein concn of determining by the requirement of constructed tissue; Under ice bath, make gel alkalization regulator solution with 0.1NNaOH and 0.01 NaOH solution, the pH value of xanthan gel liquid is adjusted to 7.2-7.5; The nutrient solution and the serum of synthetic neutrophilous organism coagulant liquid, 10 times of concentration are mixed in 8: 1: 1 ratios, obtain the pre-state xanthan gel that coagulates; Volume according to constructed tissue, ask for the pre-xanthan gel and the viable cell of coagulating of aequum respectively, dress viable cell and not having in the 50mL centrifuge tube of nutrient solution in gel joined fully is mixed with dropper piping and druming under the ice bath, obtains cell-xanthan gel suspension at last.
6, make up the method for engineering tissue according to claim 1, it is characterized in that the inoculation of said cell-xanthan gel suspension and organizational project three-dimensional rack is that the sterilization or the organizational project three-dimensional stent material of aquation are placed culture dish, cell-xanthan gel suspension is dripped in the organizational project three-dimensional stent material, the cell one biological gel suspension to material reaches capacity again; The culture dish that forms cell one gel one support complex body was inserted in 37 ℃, 5%CO2 incubator 10-15 minute; Add the desired nutrient solution of tissue culture, and break away from the bottom of making integration engineering tissue block and culture dish, all change nutrient solution after 3-5 hour and continue to cultivate.
7, make up the method for engineering tissue according to claim 1, the cultivation that it is characterized in that said tissue block is that tissue block is inserted in the culture dish, add desired nutrient solution, again culture dish is inserted in 37 ℃, 5%CO2 incubator and cultivate, changed nutrient solution once in per two days, to obtaining sophisticated tissue block.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314460C (en) * | 2004-07-15 | 2007-05-09 | 浙江大学 | Method for compounding aquo-gel to porous tissue engineering rack |
| CN102614547A (en) * | 2012-03-13 | 2012-08-01 | 中山大学中山眼科中心 | Method for rapidly constructing multilayer cells |
| CN104027845A (en) * | 2014-06-09 | 2014-09-10 | 吉林大学第二医院 | Method for constructing tissue engineering compound in vitro |
| CN106693057A (en) * | 2016-11-15 | 2017-05-24 | 广西医科大学 | Cell-biological scaffold complex and 3D printing forming method thereof |
| CN107366331A (en) * | 2017-08-29 | 2017-11-21 | 朱学斌 | A kind of auto-pneumatic cylinder |
| CN107400661A (en) * | 2017-06-29 | 2017-11-28 | 武汉枫霖科技有限公司 | A kind of method of the three-dimensional liver cancer tissue structure based on biological 3D printing hydrogel |
| CN107737373A (en) * | 2017-12-07 | 2018-02-27 | 山东隽秀生物科技股份有限公司 | A kind of preparation method of tendon repair material |
| CN115369065A (en) * | 2022-08-09 | 2022-11-22 | 华南理工大学 | Three-dimensional cell culture scaffold based on hydrogel and preparation method thereof |
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2002
- 2002-07-04 CN CNB021334013A patent/CN1177041C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314460C (en) * | 2004-07-15 | 2007-05-09 | 浙江大学 | Method for compounding aquo-gel to porous tissue engineering rack |
| CN102614547A (en) * | 2012-03-13 | 2012-08-01 | 中山大学中山眼科中心 | Method for rapidly constructing multilayer cells |
| CN102614547B (en) * | 2012-03-13 | 2015-04-15 | 中山大学中山眼科中心 | Method for rapidly constructing multilayer cells |
| CN104027845A (en) * | 2014-06-09 | 2014-09-10 | 吉林大学第二医院 | Method for constructing tissue engineering compound in vitro |
| CN104027845B (en) * | 2014-06-09 | 2015-11-18 | 吉林大学第二医院 | A kind of method of external structure organizational project complex |
| CN106693057A (en) * | 2016-11-15 | 2017-05-24 | 广西医科大学 | Cell-biological scaffold complex and 3D printing forming method thereof |
| CN107400661A (en) * | 2017-06-29 | 2017-11-28 | 武汉枫霖科技有限公司 | A kind of method of the three-dimensional liver cancer tissue structure based on biological 3D printing hydrogel |
| CN107366331A (en) * | 2017-08-29 | 2017-11-21 | 朱学斌 | A kind of auto-pneumatic cylinder |
| CN107737373A (en) * | 2017-12-07 | 2018-02-27 | 山东隽秀生物科技股份有限公司 | A kind of preparation method of tendon repair material |
| CN115369065A (en) * | 2022-08-09 | 2022-11-22 | 华南理工大学 | Three-dimensional cell culture scaffold based on hydrogel and preparation method thereof |
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