阅读说明：本技术 一种规模化制备人类细胞外基质筛选培养方法 (Screening and culturing method for large-scale preparation of human extracellular matrix ) 是由 沈政 程顺巧 江振华 于 2020-12-30 设计创作，主要内容包括：本发明公开了一种规模化制备人类细胞外基质筛选培养方法,S1、材料取样：对人体的皮肤表面进行消毒杀菌,从中取样带有细胞经脱处理的皮肤,并对该细胞进行低温的保存,待需要使用时取出；S2、基质分离：利用冷酶交联剂法进行脱细胞处理,本发明涉及细胞培养技术领域。该规模化制备人类细胞外基质筛选培养方法,通过对细胞外基质的取样,并进行外界的环境模拟试验操作,并进行比对后,不仅在操作的过程中避免细胞或是细胞外基质受到外界的细菌等影响,而且可以使得操作更加的简单化,同时在进行实验后,更能选用出相对性价比较高的环境方案,从而实现了自动规模化的生产制备,而且极大的提高了细胞外基质培养成功的存活率。(The invention discloses a screening and culturing method for preparing human extracellular matrix in large scale, which comprises the following steps of S1: sterilizing the skin surface of a human body, sampling skin with cells subjected to removal treatment, storing the cells at low temperature, and taking out the cells when in use; s2, matrix separation: the invention relates to the technical field of cell culture, and discloses a method for carrying out cell removal treatment by using a cold enzyme cross-linking agent method. According to the screening and culturing method for preparing the human extracellular matrix on a large scale, the extracellular matrix is sampled, external environment simulation test operation is carried out, and after comparison, influence of cells or the extracellular matrix on external bacteria and the like is avoided in the operation process, operation can be simplified, and meanwhile, an environment scheme with higher relative cost performance can be selected after experiment, so that automatic large-scale production and preparation are realized, and the survival rate of successful culture of the extracellular matrix is greatly improved.)

1. A screening and culturing method for preparing human extracellular matrix in a large scale is characterized in that: the method specifically comprises the following steps:

s1, material sampling: sterilizing the skin surface of a human body, sampling skin with cells subjected to removal treatment, storing the cells at low temperature, and taking out the cells when in use;

s2, matrix separation: carrying out decellularization treatment by using a cold enzyme cross-linking agent method, then treating cells by digestion, wall breaking and centrifugation of the cells, generating cell suspension after standing for a period of time, then separating and cleaning the cell suspension to obtain the required extracellular matrix under the inner wall of the cell skin, and storing the obtained extracellular matrix;

s3, culture test: taking out 9 equal parts with the same volume from the obtained extracellular matrix, respectively placing the parts in 9 corresponding culture dishes, adding the same culture solution into the culture dishes, performing amplification culture by using the culture solution, placing the culture dishes in different environments for culturing for different durations, and observing the change of an observer in the culture process;

s4, activity detection: and preferentially detecting the culture dish after the corresponding time is finished, extracting the culture dish through the sterilized and disinfected extraction tube, putting the extracellular matrix solution in the extraction tube into a detection kit for detection, and obtaining corresponding results for comparison.

2. The screening culture method for large-scale preparation of human extracellular matrix according to claim 1, wherein the screening culture method comprises the following steps: the skin tissue cells of the human skin are taken in the S1 and are stored in a storage box at 5 ℃.

3. The screening culture method for large-scale preparation of human extracellular matrix according to claim 1, wherein the screening culture method comprises the following steps: the cold enzyme cross-linking agent method in S2 is to utilize bifunctional or multifunctional reagents to carry out cross-linking reaction among enzyme molecules, between the enzyme molecules and inert protein or between the enzyme molecules and carriers to form covalent bond connection so as to prepare the immobilized enzyme.

4. The screening culture method for large-scale preparation of human extracellular matrix according to claim 1, wherein the screening culture method comprises the following steps: and in the step S2, the digestion of the cells is stopped by using the culture solution, the cells are broken by using a wall breaking machine, the cells after the wall breaking are centrifuged by using a centrifuge, the centrifugation speed is 15Kr/min, the operation is repeated for 2-3 times, and then the cells are kept still.

5. The screening culture method for large-scale preparation of human extracellular matrix according to claim 1, wherein the screening culture method comprises the following steps: and filtering the cell suspension in the S2 to separate the cell suspension into clear liquid and cell integument, collecting the cell integument, extracting extracellular matrix under the inner wall of the cell integument and storing the extracellular matrix.

6. The screening culture method for large-scale preparation of human extracellular matrix according to claim 1, wherein the screening culture method comprises the following steps: the 9 aliquots of extracellular matrix taken in S3 were all 0.25mg, and the culture solution added to the culture dish was all aliquots of 100ml solution, and amplification culture was performed.

7. The screening culture method for large-scale preparation of human extracellular matrix according to claim 1, wherein the screening culture method comprises the following steps: the environments tested in S3 are: experimental environment 1: culturing at 5 ℃, wherein the culturing time is 1h, 2h and 5h respectively; experimental environment 2: culturing at 20 ℃, wherein the culture time is 1h, 2h and 5h respectively; experimental environment 3: culturing at 37 ℃ for 1h, 2h and 5h respectively.

8. The screening culture method for large-scale preparation of human extracellular matrix according to claim 1, wherein the screening culture method comprises the following steps: the extraction tube adopted in the S4 is sterilized at high temperature, the extraction tube is not reused, and the extraction capacity is 5ml for detection.

Technical Field

The invention relates to the technical field of cells, in particular to a screening culture method for preparing human extracellular matrix in a large scale.

Background

In multicellular organisms, a complex network of many macromolecules, called the extracellular matrix, surrounds the cells; the extracellular matrix is mainly composed of 5 types of substances, namely collagen, non-collagen, elastin, proteoglycan and aminoglycan, which is a basement membrane at the base of epithelial or endothelial cells and a mesenchymal connective tissue at the cell-cell adhesion structure, and epithelial cells develop along the basal layer during embryonic development or callus regeneration, so that several information regulating cell growth and development is transmitted through the extracellular matrix.

According to the patent names: a clinical treatment grade epidermal stem cell for cell treatment is prepared by applying human extracellular matrix screening and large-scale culture, and the patent publication number is as follows: CN104312970A, wherein the treatment operations including digestion, cleaning and the like of cells by using three culture solutions are adopted to obtain corresponding experimental results, immunogenic cells are screened out to safely obtain immune escape, the transplantation time is prolonged, the tissue engineering clinical application is increased, and a solution of absolute advantage clinical treatment level cells is provided for promoting wound healing and skin diseases.

However, the existing large-scale preparation has the following problems that the corresponding activity of the cells is different, the corresponding activity degree of the cells cannot be maintained for a long time, in addition, in the culture process, more external factors are applied, and the survival rate of the cultured cells is lower, so the invention provides the method for screening and culturing the human extracellular matrix in a large-scale manner.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a screening and culturing method for preparing human extracellular matrix in a large scale, and solves the problems that the existing large-scale preparation can not maintain the cell activity degree for a long time and the survival rate is lower.

In order to achieve the purpose, the invention is realized by the following technical scheme: a screening and culturing method for preparing human extracellular matrix in a large scale specifically comprises the following steps:

s1, material sampling: sterilizing the skin surface of a human body, sampling skin with cells subjected to removal treatment, storing the cells at low temperature, and taking out the cells when in use;

s2, matrix separation: carrying out decellularization treatment by using a cold enzyme cross-linking agent method, then treating cells by digestion, wall breaking and centrifugation of the cells, generating cell suspension after standing for a period of time, then separating and cleaning the cell suspension to obtain the required extracellular matrix under the inner wall of the cell skin, and storing the obtained extracellular matrix;

s3, culture test: taking out 9 equal parts with the same volume from the obtained extracellular matrix, respectively placing the parts in 9 corresponding culture dishes, adding the same culture solution into the culture dishes, performing amplification culture by using the culture solution, placing the culture dishes in different environments for culturing for different durations, and observing the change of an observer in the culture process;

s4, activity detection: and preferentially detecting the culture dish after the corresponding time is finished, extracting the culture dish through the sterilized and disinfected extraction tube, putting the extracellular matrix solution in the extraction tube into a detection kit for detection, and obtaining corresponding results for comparison.

Preferably, the skin tissue cells of human skin are taken in S1 and stored in a storage box at 5 ℃.

Preferably, the cold enzyme cross-linking agent method in S2 is to prepare an immobilized enzyme by using a bifunctional or multifunctional reagent to perform a cross-linking reaction between enzyme molecules, between an enzyme molecule and an inert protein, or between an enzyme molecule and a carrier, thereby forming a covalent bond linkage.

Preferably, in S2, the digestion of the cells is terminated by using the culture solution, the cells are broken by using a wall breaking machine, the cells after being broken are centrifuged by using a centrifuge at a centrifugation speed of 15Kr/min for 2-3 times, and then the cells are left to stand still.

Preferably, the cell suspension in S2 is filtered to separate into clear liquid and cell outer skin, the cell outer skin is collected, and the extracellular matrix under the inner wall of the cell outer skin is extracted and stored.

Preferably, the 9 equal parts of extracellular matrix taken out in S3 are all 0.25mg, and the culture solution added to the culture dish is all equal parts of 100ml solution, and amplification culture is carried out.

Preferably, the environments tested in S3 are: experimental environment 1: culturing at 5 ℃, wherein the culturing time is 1h, 2h and 5h respectively; experimental environment 2: culturing at 20 ℃, wherein the culture time is 1h, 2h and 5h respectively; experimental environment 3: culturing at 37 ℃ for 1h, 2h and 5h respectively.

Preferably, the extraction tube used in S4 is sterilized by high temperature sterilization, and the extraction tube is not reused, and the extraction volume is 5ml for detection.

Advantageous effects

The invention provides a screening and culturing method for preparing human extracellular matrix in a large scale. Compared with the prior art, the method has the following beneficial effects: the screening and culturing method for preparing the human extracellular matrix in a large scale comprises the following steps of S1, material sampling: sterilizing the skin surface of a human body, sampling skin with cells subjected to removal treatment, storing the cells at low temperature, and taking out the cells when in use; s2, matrix separation: carrying out decellularization treatment by using a cold enzyme cross-linking agent method, then treating cells by digestion, wall breaking and centrifugation of the cells, generating cell suspension after standing for a period of time, then separating and cleaning the cell suspension to obtain the required extracellular matrix under the inner wall of the cell skin, and storing the obtained extracellular matrix; s3, culture test: taking out 9 equal parts with the same volume from the obtained extracellular matrix, respectively placing the parts in 9 corresponding culture dishes, adding the same culture solution into the culture dishes, performing amplification culture by using the culture solution, placing the culture dishes in different environments for culturing for different durations, and observing the change of an observer in the culture process; s4, activity detection: preferentially detecting the culture dish after the completion of the corresponding time, extracting the culture dish through the sterilized extraction tube, placing the extracellular matrix solution in the extraction tube into a detection kit for detection, obtaining corresponding results for comparison, placing skin tissue cells of human skin taken in S1 into a storage box at 5 ℃ for storage, performing crosslinking reaction among enzyme molecules, between the enzyme molecules and inert protein or between the enzyme molecules and a carrier by using a bifunctional or multifunctional reagent to form covalent bond connection to prepare immobilized enzyme in S2, terminating the digestion of the cells by using a culture solution in S2, breaking the walls of the cells by using a wall breaking machine, centrifuging the cells after the wall breaking by using a centrifuge at the centrifuging speed of 15Kr/min, and repeating the operation for 2-3 times, then standing, filtering the cell suspension in S2 to separate into clear liquid and cell skin, collecting the cell skin, extracting the extracellular matrix under the inner wall of the cell skin, and storing, wherein 0.25mg of the 9 equal parts of the extracellular matrix taken out in S3 are all equal parts of 100ml solution added into a culture dish for amplification culture, and the experimental environments in S3 are respectively as follows: experimental environment 1: culturing at 5 ℃, wherein the culturing time is 1h, 2h and 5h respectively; experimental environment 2: culturing at 20 ℃, wherein the culture time is 1h, 2h and 5h respectively; experimental environment 3: cultivate under 37 degrees centigrade, and the time length of cultivation is 1h respectively, 2h and 5h, the extraction tube that adopts in S4 passes through pasteurization disinfection, and the extraction tube does not carry out used repeatedly, the capacity of simultaneously extracting is 5ml and detects, through the sample to extracellular matrix, and outside environmental simulation test operation carries out, and compare the back, not only avoid the cell or extracellular matrix to receive influence such as outside bacterium at the in-process of operation, and can make the simplification more of operation, simultaneously after carrying out the experiment, more can choose the higher environmental scheme of relative sexual valence for use, thereby automatic large-scale production preparation has been realized, and very big improvement the successful survival rate of extracellular matrix cultivation.

Drawings

FIG. 1 is a flow chart of a culture method of the present invention;

FIG. 2 is a table comparing the results of activity assays of the present invention;

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-2, the present invention provides a technical solution: a screening and culturing method for preparing human extracellular matrix in a large scale specifically comprises the following steps:

s1, material sampling: sterilizing the skin surface of a human body, sampling skin with cells subjected to removal treatment, storing the cells at low temperature, and taking out the cells when in use;

s2, matrix separation: carrying out decellularization treatment by using a cold enzyme cross-linking agent method, then treating cells by digestion, wall breaking and centrifugation of the cells, generating cell suspension after standing for a period of time, then separating and cleaning the cell suspension to obtain the required extracellular matrix under the inner wall of the cell skin, and storing the obtained extracellular matrix;

s3, culture test: taking out 9 equal parts with the same volume from the obtained extracellular matrix, respectively placing the parts in 9 corresponding culture dishes, adding the same culture solution into the culture dishes, performing amplification culture by using the culture solution, placing the culture dishes in different environments for culturing for different durations, and observing the change of an observer in the culture process;

s4, activity detection: and preferentially detecting the culture dish after the corresponding time is finished, extracting the culture dish through the sterilized and disinfected extraction tube, putting the extracellular matrix solution in the extraction tube into a detection kit for detection, and obtaining corresponding results for comparison.

Preferably, the skin tissue cells of human skin are taken in S1 and stored in a storage box at 5 degrees celsius.

In the embodiment of the invention, the cold enzyme cross-linking agent method in S2 is to utilize bifunctional or multifunctional reagents to carry out cross-linking reaction among enzyme molecules, between the enzyme molecules and inert protein or between the enzyme molecules and a carrier to form covalent bond connection so as to prepare the immobilized enzyme.

In the embodiment of the invention, in S2, the digestion of the cells is terminated by using the culture solution, the cells are broken by using a wall breaking machine, the cells after being broken are centrifuged by using a centrifuge at a centrifugation speed of 15Kr/min for 2-3 times, and then the cells are kept still.

In the embodiment of the invention, the cell suspension in the S2 is filtered and divided into clear liquid and cell integument, the cell integument is collected, and extracellular matrix under the inner wall of the cell integument is extracted and stored.

In the present example, 0.25mg of extracellular matrix was removed in 9 aliquots in S3, and amplification culture was performed in 100ml aliquots of culture medium added to the petri dish.

In the embodiment of the present invention, the environments tested in S3 are: experimental environment 1: culturing at 5 ℃, wherein the culturing time is 1h, 2h and 5h respectively; experimental environment 2: culturing at 20 ℃, wherein the culture time is 1h, 2h and 5h respectively; experimental environment 3: culturing at 37 ℃ for 1h, 2h and 5h respectively.

In the embodiment of the invention, the extraction tube adopted in S4 is sterilized at high temperature, the extraction tube is not reused, and the extraction capacity is 5ml for detection.

Comparative experiment

In the above-mentioned culture experiment of S3, 9 dishes containing the same culture were placed at ambient temperatures of 5 degrees celsius, 20 degrees celsius, and 37 degrees celsius for 1 hour, 2 hours, and 5 hours, respectively, and the activity index detected by the reagent cartridge was obtained, as shown in fig. 2, the product in the dish was cultured at 20 degrees celsius for 2 hours with the highest activity value, which is an optimal culture protocol, and the rest of the dishes were cultured, and the relative activity index was low.

In conclusion, by sampling the extracellular matrix, carrying out external environment simulation test operation and comparing, the influence of external bacteria and the like on the cell or the extracellular matrix is avoided in the operation process, the operation is simplified, and meanwhile, after the experiment, an environment scheme with higher relative cost performance can be selected, so that the automatic large-scale production and preparation are realized, and the survival rate of successful culture of the extracellular matrix is greatly improved.

And those not described in detail in this specification are well within the skill of those in the art.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.