CN101735984B - Simple and convenient method for inoculating cell for scaffold material of tissue engineering - Google Patents
Simple and convenient method for inoculating cell for scaffold material of tissue engineering Download PDFInfo
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- CN101735984B CN101735984B CN2008101477008A CN200810147700A CN101735984B CN 101735984 B CN101735984 B CN 101735984B CN 2008101477008 A CN2008101477008 A CN 2008101477008A CN 200810147700 A CN200810147700 A CN 200810147700A CN 101735984 B CN101735984 B CN 101735984B
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Abstract
The invention provides a simple, convenient and effective method for realizing three-dimensional uniform inoculation of seed cells on a porous scaffold material of biological engineering. The method is characterized by comprising the following steps: using a clinically conventional sterilized syringe as an inoculating tool; putting a required biological scaffold material into the syringe from the back part thereof; sucking cell suspension, required for inoculating, with a corresponding number of cells; preserving a certain amount of air in the syringe; sealing the opening of the syringe; withdrawing the syringe; and enabling the cell suspension to permeate into air gaps of a porous material by using a formed negative pressure so as to realize the initial-stage uniform distribution and inoculation of the seed cells in the scaffold material. Compared with the method for realizing the initial-stage three-dimensional uniform inoculation of the scaffold material in the conventional tissue engineering practice, the method of the invention has the advantages of simplicity, convenience, easy implementation, and capability of achieving the effect of initial-stage three-dimensional uniform inoculation similar to special equipment without purchasing a special instrument with a large amount of funds. The inoculated cells are not damaged because the acting time of the negative pressure is short and the pressure is low.
Description
Technical field
The present invention relates in a kind of organizational project in vitro study and the clinical application method of the even cell inoculation of realization initial stage two dimension on porous support materials.
Background technology
In the organizational project practice, some need cultivate amplification with seed cell under the environment of external two dimension, reaching certain number, or passes through the directed differentiation of the intervening measure realization cell in culturing process, is inoculated on the timbering material then; Some is then gathering after the seed cell direct inoculation on timbering material.The cell inoculation process in the early stage the cell two-dimensional space distribute with and subsequent organizational project cultivate in the atomization and playing the part of very important role.It is one of important evaluation index of cell inoculation method that primary stage of inoculation reaches the uniform distribution of cell in timbering material.
The inoculation method that uses comprises static inoculation method and dynamic inoculation method at present.Wherein, static inoculation is exactly directly cell suspension to be dripped on timbering material, allows cell suspension enter timbering material inside by osmosis.This method is inoculation method relatively more commonly used at present, but can't realizes the uniform distribution of cell on three-dimensional structure owing to easy and simple to handle.More common dynamic inoculation method has bio-reactor method, centrifuging, vacuum machine method, revolving bottle method etc. at present, and these dynamic inoculation methods can reach the uniform distribution of incipient cell on how empty stock support three-dimensional space.But these methods all need the instrument of special costliness to cooperate, and operation is complicated.Therefore, press for the equally distributed cell inoculation method on a kind of new simple and easy to do realization cell inoculation initial stage three-dimensional space of developing in this area.
Summary of the invention
The present invention is intended to utilize laboratory and clinical equipment configuration commonly used, finish the initial stage of seed cell on biologic bracket material simple and effective evenly inoculates.And in the cell inoculation process, adopt the shortest intervention time and minimum intervention intensity, farthest keep the biologic activity of cell.
The invention provides a kind of simple and effective ground method to achieve these goals, its step is as follows:
A. be ready to the timbering material of required inoculating cell, and select corresponding clinical disposable syringe specification commonly used according to the size of timbering material.
B. ready timbering material is put into from the syringe rear portion, put back to the syringe piston post, release unnecessary air.
C. suck cell suspension from injector orifice, the cell suspension amount is as the criterion just to flood timbering material.The cell concn of cell suspension is according to the different vaccination purpose, and concentration is different.
D. release unnecessary air, in syringe, except that support cell suspension complex body, only keep the 1ml air.
E. block syringe port, the pumpback piston boit makes the interior air of syringe cylinder be increased to 3ml, and kept for 5 seconds under this state.Secondary is kept in pumpback so repeatedly.
F. extract piston boit, pour out unnecessary cell suspension, take out timbering material and place in the culture dish.At 37 ℃, contain in the 5%CO2 cell cultures incubator and left standstill 2 hours, treat that cell pastes a pair back fully and adds required substratum.Perhaps after inoculation, be filled directly into the operation defect.
Description of drawings
Figure one conventional static cell inoculation mode drips cell suspension in porosity to be 60%TCP timbering material after last two hour, to carry out MTT dyeing observation of cell and distribute.As seen active cells mainly is deposited in the love of adding cell suspension and expects a side.(MTT can act on the respiratory chain in the viable cell plastosome, generates the blue crystallization of formazan under the effect of succinodehydrogenase and cytochrome C.Make have active cell under naked eyes as seen.)
Figure two adopts method inoculating cell of the present invention on identical TCP timbering material, carries out MTT dyeing observation of cell after two hours and distributes.Visible cell is evenly distributed in the material.
Figure three static inoculation TCP timbering materials are cultivated 2 all rear center position stereoscan photographs, do not have cell survival.(×200)
Figure four adopts the present invention to inoculate the TCP timbering material and cultivates 2 all rear center position stereoscan photographs, the visible cell survival.(×200)
Embodiment
Embodiment 1:
Human bone marrow stroma stem cell is seeded in and carries out osteogenic induction on the TCP timbering material.
The myeloid tissue of hospital clinical collection places on the Percoll liquid level of equivalent centrifugal 30 minutes of 3000rpm after the dilution of PBS liquid.Gather the mononuclear cell layer of oyster white muddiness and cultivate amplification.Being cultured to and adjusting cell suspension after the s-generation cell is 5 * 10
6/ ml prepares the inoculation support.
Preparation TCP timbering material is 5mm * 5mm * 4mm size, behind the sterilization and drying three timbering materials is put into 10ml syringe inside.Suck the 1ml cell suspension, and reserve the 1ml air in syringe inside.
Block injector orifice, the pumpback piston boit makes the syringe inner air reach 3ml, keeps and well slowly recovers pressure 5 seconds.Three times so repeatedly.Unnecessary cell suspension reclaimed continue next time to use, the cytoskeleton complex body that inoculation is good moves into culture dish, in cell culture incubator, leave standstill 2 hours after, add the osteogenic induction substratum.
Embodiment 2:
Human bone marrow stroma stem cell is seeded on the TCP timbering material and is carried out to chondrocyte induction.
The myeloid tissue that hospital gathers places on the Percoll liquid level of equivalent centrifugal 30 minutes of 3000rpm after the dilution of PBS liquid.Gather the mononuclear cell layer of oyster white muddiness and cultivate amplification.Being cultured to and adjusting cell suspension after the s-generation cell is 14 * 10
6/ ml prepares the inoculation support.
Preparation TCP timbering material is 5mm * 5mm * 4mm size, behind the sterilization and drying three timbering materials is put into 10ml syringe inside.Suck the 1ml cell suspension, and reserve the 1ml air in syringe inside.
Block injector orifice, the pumpback piston boit makes the syringe inner air reach 3ml, keeps and well slowly recovers pressure 5 seconds.Three times so repeatedly.Unnecessary cell suspension reclaimed continue next time to use, the cytoskeleton complex body that inoculation is good moves into culture dish, in cell culture incubator, leave standstill 2 hours after, add into the chondrocyte induction substratum.
Embodiment 3:
MC3T3-E1 cell inoculation carries out the osteogenic induction experiment on the PDLLA support.
Vitro culture MC3T3-E1 clone, adjusting cell suspension behind enough numbers is 5 * 10
6/ ml prepares the inoculation support.
Preparation PDLLA timbering material is 10mm * 10mm * 5mm size, behind the sterilization and drying three timbering materials is put into 10ml syringe inside.Suck the 2ml cell suspension, and reserve the 1ml air in syringe inside.
Block injector orifice, the pumpback piston boit makes the syringe inner air reach 3ml, keeps and well slowly recovers pressure 5 seconds.Secondary so repeatedly.Unnecessary cell suspension reclaimed continue next time to use, the cytoskeleton complex body that inoculation is good moves into culture dish, in cell culture incubator, leave standstill 2 hours after, add the osteogenic induction substratum.
Embodiment 4:
The human osteoblast cell is seeded on the TCP support, the interaction of research scleroblast and TCP timbering material.
People's spongy bone that hospital clinical is gathered adopts digestion method collector scleroblast, cultivates in external two-dimensional state.Collect the 3-5 generation cell, adjusting cell suspension is 5 * 10
6/ ml prepares the inoculation support.
Preparation TCP timbering material is 5mm * 5mm * 4mm size, behind the sterilization and drying three timbering materials is put into 10ml syringe inside.Suck the 1ml cell suspension, and reserve the 1ml air in syringe inside.
Block injector orifice, the pumpback piston boit makes the syringe inner air reach 3ml, keeps and well slowly recovers pressure 5 seconds.Three times so repeatedly.Unnecessary cell suspension reclaimed continue next time to use, the cytoskeleton complex body that inoculation is good moves into culture dish, in cell culture incubator, leave standstill 2 hours after, add the osteogenic induction substratum.
Embodiment 5:
The pig chondrocyte is seeded in dimensional culture on the PDLLA support, observes the effect of mechanical stress to the chondrocyte of external dimensional culture.
Gather 6 monthly age pig knee joint surface cartilages, use digestion method to collect the pig chondrocyte.In order to prevent the chondrocyte in external degeneration, collect s-generation chondrocyte, adjusting cell suspension is 14 * 10
6/ ml prepares the inoculation support.
Preparation PDLLA timbering material is 10mm * 10mm * 5mm size, behind the sterilization and drying three timbering materials is put into 10ml syringe inside.Suck the 2ml cell suspension, and reserve the 1ml air in syringe inside.
Block injector orifice, the pumpback piston boit makes the syringe inner air reach 3ml, keeps and well slowly recovers pressure 5 seconds.Secondary so repeatedly.Unnecessary cell suspension reclaimed continue next time to use, the cytoskeleton complex body that inoculation is good moves into culture dish, in cell culture incubator, leave standstill 2 hours after, add the chondrocyte induction culture medium culturing that contains TGF-β.According to test design, applying low frequency ultrasound stimulates subsequently.
Claims (2)
1. a biomaterial is realized the three-dimensional evenly method of inoculating cell of initial stage, and its step comprises:
1) cell of the required inoculation that separation and Culture is enough in earlier stage, and, adjust the concentration of cell suspension that is fit to according to the inoculation purpose, and be ready to the timbering material of required inoculating cell, it is stand-by to sterilize;
2) select corresponding inoculating tool according to the size of timbering material: the specification of asepsis injector;
3) extract syringe piston after, ready timbering material is put into from the syringe rear portion, put back to the syringe piston post, release unnecessary air;
4) suck ready cell suspension from injector orifice, the piston boit of pumpback simultaneously, the cell suspension amount is as the criterion just to flood timbering material, releases unnecessary air, in syringe, only keeps the 1ml air except that support cell suspension complex body;
5) block syringe port, the pumpback piston boit makes the interior air of syringe cylinder be increased to 3ml, and kept for 5 seconds under this state, and pumpback is kept three times so repeatedly;
6) extract piston boit, pour out unnecessary cell suspension, take out timbering material and place in the culture dish,, contain 5%CO at 37 ℃
2Left standstill in the cell cultures incubator 2 hours, and treated that cell attached the back fully and adds required substratum.
2. realize the three-dimensional evenly method of inoculating cell of initial stage according to right 1 described biomaterial, it is characterized in that: asepsis injector uses clinical disposable syringe commonly used or sterilization glass syringe.
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| CN2008101477008A CN101735984B (en) | 2008-11-27 | 2008-11-27 | Simple and convenient method for inoculating cell for scaffold material of tissue engineering |
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| CN2008101477008A CN101735984B (en) | 2008-11-27 | 2008-11-27 | Simple and convenient method for inoculating cell for scaffold material of tissue engineering |
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| CN102174397B (en) * | 2011-03-07 | 2013-08-07 | 四川大学 | Bionic three-dimensional fluid shear stress cell culture device and shear stress loading method thereof |
| CN103952296B (en) * | 2014-01-14 | 2018-11-30 | 卢建熙 | Multi-functional therapeutic treatment substance bluk recombination device |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1542121A (en) * | 2003-04-29 | 2004-11-03 | 卢建熙 | 3D dynamic input type tissue reaction device |
| CN101294131A (en) * | 2007-04-27 | 2008-10-29 | 中国药品生物制品检定所 | Bioreactor for vascellum tissue engineering |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1542121A (en) * | 2003-04-29 | 2004-11-03 | 卢建熙 | 3D dynamic input type tissue reaction device |
| CN101294131A (en) * | 2007-04-27 | 2008-10-29 | 中国药品生物制品检定所 | Bioreactor for vascellum tissue engineering |
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