CN109897823A - A method of tissue engineering comea edge is prepared based on Matrigel - Google Patents
A method of tissue engineering comea edge is prepared based on Matrigel Download PDFInfo
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- CN109897823A CN109897823A CN201910094620.9A CN201910094620A CN109897823A CN 109897823 A CN109897823 A CN 109897823A CN 201910094620 A CN201910094620 A CN 201910094620A CN 109897823 A CN109897823 A CN 109897823A
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- matrigel
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Abstract
The invention belongs to organizational project correlative technology fields, and disclose a kind of method for preparing tissue engineering comea edge based on Matrigel.This method comprises: ice hockey or dry ice pellets by Matrigel freeze thawing, are then added thereto and are stirred acquisition solidliquid mixture by (a)；(b) by the Matrigel plastic in solidliquid mixture, should during, ice hockey or dry ice pellets in solidliquid mixture are melted in the Matrigel after plastic and leave hole, so that the Matrigel matrigel obtained has porous structure；(c) limbal stem cell and corneal limbus microenvironment cell are planted in the Matrigel matrigel with porous structure of acquisition, it is made to form the tissue engineering comea edge needed for three-dimensional structure obtains in porous structure.It is simple that the present invention has a process, easy to implement, and process conditions and Matrigel porous structure are convenient for advantages such as controls.
Description
Technical field
The invention belongs to organizational project correlative technology fields, prepare tissue based on Matrigel more particularly, to one kind
The method of engineering cornea edge.
Background technique
Limbal stem cell transplantation is the primary treatments of current limbal stem cell decompensation (LSCD), and main points exist
In that need to transplant sufficient amount of limbal stem cell, patient's vision is unable to get long-term improvement if quantity is excessively few, for eyes
For the patient of illness, it is difficult to get sufficient amount of limbal stem cell from self cornea.This is just needed from self, of the same race
The sources such as allosome, relatives or corpse fetching portion limbal stem cell, expands in vitro, uses various delivery apparatus culture corneas
Then limbal stem cell returns plant patient's body again.If provide one allow limbal stem cell can preferably grow and with human body angle
The quality of graft can be improved then to obtain more preferably therapeutic effect in the similar three-dimensional structure of film edge.
Currently, the researchers of organizational project are using amnion, mucous membrane of mouth, conjunctiva etc. as limbal stem cell culture
Matrix simultaneously achieves certain success.In addition to this, tissue engineering comea edge is made using 3D printing, hydrogel, electrostatic spinning
Research it is also more and more.However, the structure of a 3D is hardly resulted in using flat films such as amnions as substrate culture cell,
The wiener structure that hydrogel has, which has, has regulation difficulty.The fiber of 3D printing is micron order fiber and is difficult to carry out essence
Micro-nano structure is made in fine tuning control, and the film hole of electrostatic spinning preparation is Nano grade, and hole is smaller, is hardly formed 3D structure.
Extracellular matrix (extra cellular matrix, ECM) is during many decision cell fates and behavior
It plays an important role.Typical extracellular matrix protein includes laminin, collagen, glycoprotein, proteoglycans.
The main function of ECM is the growth for supporting various types of cells.The growth in vitro matrix of cell can be various materials, such as pass through
Cross the protein layer of chemically treated culture dish plastics, deposition.These matrix are usually very simple, by collagen and layer adhesion
The purifying proteins such as albumen mix.This only matrix containing main ECM protein is not particularly suited for all cell types.From work
For the ECM mixture extracted in cell commonly used in cultivating the cell more sensitive to condition of culture, this may be to close due to containing
The growth factor and cell factor of key.The Matrigel being commercialized widely is used, and Matrigel can be maintained
The self-renewing and versatility of stem cell are the optimum substrates for cultivating stem cell.Correspondingly, this field is a kind of suitable there is developing
The technical need of method with property preferably based on Matrigel building tissue engineering comea edge.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the present invention provides one kind prepares tissue based on Matrigel
The method of engineering cornea edge, by preparing the Matrigel matrix to be formed with porous structure using Matrigel and ice hockey
Glue, so that cell, which is preferably grown in the hole of Matrigel, forms a kind of three-dimensional structure, to prepare a group weaver
Journey corneal limbus, wherein the hole in Matrigel matrigel is controlled by adjusting the diameter of ice hockey, the connection of porous structure
Property controlled by choosing different ice hockey smelting process, it is simple that this method has a process, easy to implement, process conditions and
Matrigel porous structure is convenient for the advantage of control.
To achieve the above object, it is proposed, according to the invention, provide (a) for Matrigel freeze thawing and make liquid；Prepare ice
Ball or dry ice pellets, and the ice hockey or dry ice pellets are added in the Matrigel of the liquid and are stirred, it is mixed to obtain solid-liquid with this
Close object；
(b) solidliquid mixture for obtaining step (a) heats up, so that Matrigel plastic therein, during being somebody's turn to do,
Ice hockey or dry ice pellets in the solidliquid mixture melt, and leave hole in the Matrigel after plastic, so that obtain
The Matrigel matrigel has porous structure；
(c) plantation corneal limbus is dry thin in the Matrigel matrigel with porous structure obtained in step (b)
Born of the same parents and corneal limbus microenvironment cell, are made it form three-dimensional structure in the porous structure, required tissue are obtained with this
Engineering cornea edge.
It is further preferred that the temperature of the freeze thawing is preferably no more than 4 DEG C in step (a), the freeze thawing time is preferably
2h~12h.
It is further preferred that in step (b), in the solidliquid mixture that step (a) is obtained
Matrigel plastic, by the Matrigel plastic preferably in tepidarium, wherein the temperature of the tepidarium is preferably 22 DEG C
~37 DEG C.
It is further preferred that in step (b), in the solidliquid mixture that step (a) is obtained
Matrigel plastic is preferably carried out according to the following steps:
(b1) solidliquid mixture for obtaining step (a), which is rapidly frozen, obtains solid-state frozen material；
(b2) by the solid-state frozen material ice hockey or dry ice pellets distil, hole is formed in the solid-state frozen material with this
Gap obtains the frozen material of porous structure；
(b3) by the frozen material heating plastic of the porous structure, Matrigel matrigel is obtained with this.
It is further preferred that the ice hockey for preparing is preferably standby using ion water making is spent in liquid nitrogen in step (a),
Wherein, the diameter of ice hockey is chosen according to actual needs, and dry ice pellets preferably use dry ice pulverizer that dry ice is made not
With the dry ice pellets powder of partial size.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain down and show
Beneficial effect:
1, the present invention regulates and controls the diameter of ice hockey by adjusting mixing speed by preparing ice hockey in liquid nitrogen, can be with
According to the ice hockey diameter that the design of the size of cell is suitable, finally produce suitable cell growth with porous structure
Matrigel；
2, the present invention prepares the Matrigel matrigel with porous structure by using the mode of tepidarium, due to such
Method ice hockey slowly melts, and the ice hockey of different location melts simultaneously, and liquid collects to leave hole and channel, obtains having company
The porous structure of the general character, so that the tissue engineering comea edge finally obtained has three-dimensional structure；
3, the present invention prepares the Matrigel with porous structure by using the mode for being rapidly frozen then removal ice hockey
Matrigel only leaves hole due to ice hockey rapid sublimation, so that the porous structure obtained is not connected to mutually, so that finally
The tissue engineering comea edge of acquisition has three-dimensional structure；
4, method flow provided by the invention is simple, easy to implement, and process conditions and Matrigel porous structure are convenient for control
System, the tissue engineering comea edge structure finally obtained are also easy to control.
Detailed description of the invention
Fig. 1 is the flow diagram that tissue engineering comea edge is prepared constructed by preferred embodiment according to the invention；
Fig. 2 is unconnectedness Matrigel porous structure schematic diagram constructed by preferred embodiment according to the invention；
Fig. 3 is connectivity Matrigel porous structure schematic diagram constructed by preferred embodiment according to the invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below
Not constituting a conflict with each other can be combined with each other.
Fig. 1 is the flow diagram that tissue engineering comea edge is prepared constructed by preferred embodiment according to the invention, such as
It is a kind of that the method for tissue engineering comea edge is prepared it is characterized in that, this method includes following step based on Matrigel shown in Fig. 1
It is rapid:
(1) Matrigel freeze thawing at 4 DEG C, Matrigel are a kind of matrix；
(2) deionized water is added in liquid nitrogen, using agitating device, centrifugal speed production is changed by control mixing speed
The ice hockey of different-diameter size out；Or the dry ice pellets powder of different-grain diameter is made in dry ice by dry ice pulverizer；
(3) ice hockey prepared is added in the Matrigel after freeze thawing, side edged mechanical stirring；
(4) melt ice hockey with different technique to regulate and control the connectivity of hole in Matrigel；
(5) cell is cultivated: by limbal stem cell and corneal limbus microenvironment cell seeding in having porous structure
On Matrigel.
Further, in step (1), the time of Matrigel freeze thawing at 4 DEG C is 2~12h；
Further, in step (2), by changing the mixing speed of rotor or stirring rod in the mixture of liquid nitrogen and water
The diameter of regulation gained ice hockey, speed is faster, and ice hockey diameter is smaller；
Further, in step (4), the Matrigel containing ice hockey is put into liquid nitrogen and is rapidly frozen, it is dry with freezing
Ice hockey in Matrigel by solid state sublimation, to remove ice hockey, is left hole by dry machine, and being rapidly heated to 22~37 DEG C makes
Matrige plastic again, produces the Matrigel for having not connected porous structure；
Further, in step (4), to Matrigel plastic under tepidarium, while ice hockey thawing leaves hole, makes
The porous structure Matrigel being connected to out.
The present invention is further illustrated below in conjunction with specific embodiments.
Embodiment 1
(1) the solid-state Matrigel for storing -20 DEG C takes out, and 12h under the conditions of being placed in 4 DEG C becomes liquid to Matrigel；
(2) magneton or stirring rod are put into liquid nitrogen, adjusting revolving speed is 200~800r/min to change centrifugal force, slowly
1~10ml deionized water is added into liquid nitrogen container, produces the ice hockey of different-diameter size, using filter paper by ice hockey from liquid nitrogen
In filter out, attention action is fast, and whole operation process should be completed on ice；
(3) ice hockey is slowly added in liquid Matrigel, while with glass bar mechanical stirring, above-mentioned steps should be on ice
It carries out；
(4) Matrigel containing ice hockey is put into liquid nitrogen and is rapidly frozen, it will be in Matrigel with freeze drier
Ice hockey by solid state sublimation, to remove ice hockey, leave hole, being rapidly heated makes Matrige plastic again to 22~37 DEG C, figure
2 be unconnectedness Matrigel porous structure schematic diagram constructed by preferred embodiment according to the invention, is made as shown in Figure 2
Provide the Matrigel of standby disconnected porous structure；
(5) at least 30min is irradiated using ultraviolet radiator to sterilize, be inoculated with limbal stem cell and corneal limbus microenvironment cell in
Have on the Matrigel of porous structure.
Embodiment 2
(1) the solid-state Matrigel for storing -20 DEG C takes out, and 2h under the conditions of being placed in 4 DEG C becomes liquid to Matrigel；
(2) magneton or stirring rod are put into liquid nitrogen, adjusting revolving speed is 200~800r/min to change centrifugal force, slowly
1~10ml deionized water is added into liquid nitrogen container, produces the ice hockey of different-diameter size, using filter paper by ice hockey from liquid nitrogen
In filter out, attention action is fast, and whole operation process should be completed on ice；
(3) ice hockey is slowly added in liquid Matrigel, while with glass bar mechanical stirring, above-mentioned steps should be on ice
It carries out；
(4) make Matrigel Rapid gelation under 22~37 DEG C of water-baths, while ice hockey thawing leaves hole, Fig. 3 be according to
Connectivity Matrigel porous structure schematic diagram constructed by the preferred embodiment of the present invention, produces have company as shown in Figure 3
The Matrigel of logical porous structure；
(5) at least 30min is irradiated using ultraviolet radiator to sterilize, be inoculated with limbal stem cell and corneal limbus microenvironment cell in
Have on the Matrigel of porous structure.
Embodiment 3
(1) the solid-state Matrigel for storing -20 DEG C takes out, and 10h under the conditions of being placed in 4 DEG C becomes liquid to Matrigel；
(2) dry ice is made to the dry ice pellets powder of different-grain diameter using dry ice pulverizer；
(3) dry ice pellets are slowly added in liquid Matrigel, while with glass bar mechanical stirring；
(4) make Matrigel Rapid gelation under 22~37 DEG C of water-baths, while dry ice pellets thawing leaves hole；
(5) at least 30min is irradiated using ultraviolet radiator to sterilize, be inoculated with limbal stem cell and corneal limbus microenvironment cell in
Have on the Matrigel of porous structure.
Embodiment 4
(1) the solid-state Matrigel for storing -20 DEG C takes out, and 10h under the conditions of being placed in 4 DEG C becomes liquid to Matrigel；
(2) dry ice is made to the dry ice pellets powder of different-grain diameter using dry ice pulverizer；
(3) dry ice pellets are slowly added in liquid Matrigel, while with glass bar mechanical stirring；
(4) Matrigel containing ice hockey is put into liquid nitrogen and is rapidly frozen, it will be in Matrigel with freeze drier
Ice hockey by solid state sublimation, to remove ice hockey, leave hole, being rapidly heated makes Matrige plastic again to 22~37 DEG C；
(5) at least 30min is irradiated using ultraviolet radiator to sterilize, be inoculated with limbal stem cell and corneal limbus microenvironment cell in
Have on the Matrigel of porous structure.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (5)
1. a kind of method for preparing tissue engineering comea edge based on Matrigel, which is characterized in that this method includes the following steps:
(a) Matrigel freeze thawing is made into liquid；Ice hockey or dry ice pellets are prepared, and will be described in the ice hockey or dry ice pellets addition
It is stirred in the Matrigel of liquid, solidliquid mixture is obtained with this；
(b) solidliquid mixture for obtaining step (a) heats up, so that Matrigel plastic therein, described during being somebody's turn to do
Ice hockey or dry ice pellets in solidliquid mixture melt, and leave hole in the Matrigel after plastic, so that is obtained is described
Matrigel matrigel has porous structure；
(c) in the Matrigel matrigel with porous structure obtained in the step (b) plantation limbal stem cell and
Corneal limbus microenvironment cell, makes it form three-dimensional structure in the porous structure, and required organizational project is obtained with this
Corneal limbus.
2. a kind of method for preparing tissue engineering comea edge based on Matrigel as described in claim 1, which is characterized in that
In step (a), the temperature of the freeze thawing is preferably no more than 4 DEG C, and the freeze thawing time is preferably 2h~12h.
3. a kind of method for preparing tissue engineering comea edge based on Matrigel as claimed in claim 1 or 2, feature exist
In, in step (b), the Matrigel plastic by the solidliquid mixture of step (a) acquisition, preferably in tepidarium
It is middle by the Matrigel plastic, wherein the temperature of the tepidarium is preferably 22 DEG C~37 DEG C.
4. a kind of method for preparing tissue engineering comea edge based on Matrigel as claimed in claim 1 or 2, feature exist
In, in step (b), the Matrigel plastic by the solidliquid mixture of step (a) acquisition, preferably according to following
Step carries out:
(b1) solidliquid mixture for obtaining step (a), which is rapidly frozen, obtains solid-state frozen material；
(b2) by the solid-state frozen material ice hockey or dry ice pellets distil, hole is formed in the solid-state frozen material with this,
Obtain the frozen material of porous structure；
(b3) by the frozen material heating plastic of the porous structure, Matrigel matrigel is obtained with this.
5. a kind of method for preparing tissue engineering comea edge based on Matrigel according to any one of claims 1-4, special
Sign is, described to prepare ice hockey preferably using to spend ion water making in liquid nitrogen standby, wherein the diameter of ice hockey in step (a)
Size is chosen according to actual needs, and dry ice pellets preferably use dry ice pulverizer that dry ice is made to the dry ice pellets powder of different-grain diameter
End.
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| US20170283773A1 (en) * | 2016-03-29 | 2017-10-05 | Catholic University Industry Academic Cooperation Foundation | Methods for improving proliferation and stemness of limbal stem cells |
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2019
- 2019-01-31 CN CN201910094620.9A patent/CN109897823A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101484574A (en) * | 2006-04-28 | 2009-07-15 | 雷诺维特有限公司 | Substrate for the growth of cultured cells in three dimensions |
| CN107075469A (en) * | 2014-06-27 | 2017-08-18 | 加利福尼亚大学董事会 | Mammal limbal stem cell, its production method and its purposes of culture |
| CN106543467A (en) * | 2015-09-16 | 2017-03-29 | 清华大学 | A kind of ice glue support and its production and use |
| US20170283773A1 (en) * | 2016-03-29 | 2017-10-05 | Catholic University Industry Academic Cooperation Foundation | Methods for improving proliferation and stemness of limbal stem cells |
Non-Patent Citations (3)
| Title |
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| HUA-TAO XIE 等: "Limbal Epithelial Stem/Progenitor Cells Attract Stromal Niche Cells by SDF-1/CXCR4 Signaling to Prevent Differentiation", 《STEM CELLS》 * |
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