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Method for remotely delivering recombinant amniotic culture epithelial membrane

A membrane and epithelial technology, applied in the field of cell culture, can solve problems such as poor mastery, lack of medium, cumbersome process, etc., and achieve the effect of convenient remote delivery, maintaining cell viability, and good cell viability

Active Publication Date: 2015-03-25
SHANDONG EYE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the process of mature culture system is cumbersome, and feeder cells are mostly used, and the composition of the medium is complex, which is not easy to master. Therefore, it is particularly important to safely transport the membranes with good culture properties to patients who need transplantation to improve the condition of the ocular surface
However, at present, when the recombinant amnion is transported long-distance to culture the epithelial membrane, the lack of medium often occurs, and the cells are easy to contaminate and apoptotic.

Method used

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  • Method for remotely delivering recombinant amniotic culture epithelial membrane
  • Method for remotely delivering recombinant amniotic culture epithelial membrane

Examples

Experimental program
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Effect test

preparation example Construction

[0013] 1. Preparation of three-dimensional gelatin scaffolds:

[0014] Prepare the gelatin sponge bottom pad: use a compass to draw a circle with a diameter of 32mm on the gelatin sponge, measure the height to 6mm, and cut the round pad to make the bottom pad;

[0015] Preparation of gelatin cylinder ring: use a compass to draw a circle with a diameter of 25mm on the gelatin sponge, measure the height to 10mm, cut off the cylinder, keep the wall thickness of the cylinder at 3mm, and cut off the rest with a circular stainless steel knife. Sterilize the prepared base pad and cylindrical ring with ethylene oxide for later use ( figure 1 with figure 2 ).

[0016] 2. Three-dimensional three-dimensional gelatin scaffold packaging cell membrane:

[0017] Put the sterilized gelatin sponge pad into the bottom of the 6-well culture plate, place the gelatin sponge ring on the edge of the culture well, take out the cultured cell membrane sleeve from the transwell, insert the epithelia...

Embodiment 1

[0022] After transporting for 48 hours, take out the cell membrane, add 0.25% trypsin-0.02% EDTA to half of the membrane, digest at 37°C for 10 minutes, centrifuge at 1500rpm / min for 5 minutes, add cell culture medium and centrifuge to resuspend to make a single cell suspension, take 10μl of cells Suspension, add an equal volume of 0.4% trypan blue solution, pipette and mix well, use a cell counter to count the proportion of living cells and the total cell amount, the total amount of cells is 6.26×10 5 , the proportion of living cells was 56%; the other half of the cell membranes were fixed with paraformaldehyde, embedded in paraffin, made paraffin sections, and stained with HE to observe the cell morphology and membrane structure. Attached to the homogeneous amniotic membrane surface, about 3-5 layers, the cells are closely arranged, the bottom layer cells are nearly cuboid, while the upper layer cells are relatively flat.

Embodiment 2

[0024] Prepare the three-dimensional gelatin scaffold according to the conventional method, put the sterilized gelatin sponge pad into the bottom of the 6-well culture plate, put the gelatin sponge ring on it, take out the cultured cell membrane from the transwell, and insert the epithelial side up into the gelatin sponge ring Inside, add nutrient solution to the sponge until it is saturated, cover the culture plate, seal the culture plate with a sealing film, put it in an insulated box and send it by express delivery.

[0025] In the traditional transportation method, after the cultured cell membrane is taken out, it is directly placed in a 60mm culture dish, a small amount of medium is added to cover the membrane, and the sealing film is sealed and then packed in a box for express delivery. Since the cell membrane cannot be fixed, it is very easy to cause the cell membrane or cells to fall off due to position changes during transportation, and the culture medium in the cultur...

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Abstract

The invention aims to provide a method for remotely delivering a recombinant amniotic culture epithelial membrane. The method comprises the following steps: preparing a cylindrical gelatin sponge bottom pad, and preparing a gelatin sponge cylindrical ring for containing a membrane sleeve; sterilizing the bottom pad and the cylindrical ring, putting the bottom pad and the cylindrical ring into culture holes, putting a completely cultured recombinant amniotic culture epithelial membrane sleeve into the interior of the sponge ring, adding a cell culture medium to soak a cell patch and fill gelatin sponges, sealing by using a sealing membrane, and performing remote delivery. According to the method disclosed by the invention, the gelatin sponge serves as a three-dimensional stent and highly expands after nutrient solution is added, so that the culture sleeve can be firmly fixed; the nutrient solution adsorbed in the gelatin sponge at the bottom has a good trophic action on cells, so that the cell activity is well maintained. Moreover, according to the nutrient solution adsorbed by the gelatin sponge, the cells can be kept in sterile and activated states, cell contamination caused by outside leakage of the added nutrient solution is avoided, and the remote delivery is conveniently realized.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for remotely delivering recombined amniotic membrane cultured epithelial membranes. Background technique [0002] Corneal disease is the second most common cause of blindness in my country. According to statistics, there are about 4-5 million monocular and binocular corneal blind people across the country, among which patients with limbal stem cell deficiency due to various reasons account for a considerable proportion. The lack of corneal limbal stem cells makes the corneal epithelium lose its ability to regenerate and repair, causing corneal conjunctival epithelialization, neovascularization, persistent chronic inflammation, repeated epithelial defects, stromal scarring, and corneal autolysis and ulcers. In severe cases, it can lead to blindness. For patients with loss of all limbal stem cells, autologous or allogeneic limbal stem cell transplantation...

Claims

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Application Information

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IPC IPC(8): A61L27/36A61L27/54
Inventor 周庆军王瑶段豪云杨玲玲
Owner SHANDONG EYE INST
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