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CN-121975730-A - Stem cell-based microvesicle and preparation method and application thereof

CN121975730ACN 121975730 ACN121975730 ACN 121975730ACN-121975730-A

Abstract

The invention discloses a stem cell-based microvesicle, a preparation method and application thereof, belonging to the technical field of biological tissue engineering, in particular to a preparation method of the stem cell-based microvesicle, which comprises the steps of culturing stem cells until the cell fusion degree is 80-90%, performing hypoxia treatment, and cleaning for 2-3 times; culturing the cleaned stem cells by standing, adding a calcium ion carrier, performing ultrasonic treatment, immediately absorbing a serum-free basic culture medium containing the calcium ion carrier, washing the cells by PBS, culturing for 24 hours, respectively collecting and combining cell supernatants, performing first centrifugation, removing cell fragments, second centrifugation, removing dead cells and large cell fragments, performing third centrifugation, removing organelles, performing fourth centrifugation, removing large vesicles to obtain a final supernatant, and filtering and centrifuging to obtain microvesicle sediment. The preparation method of the microvesicles aims at systematically improving the yield and the bioactivity of stem cell microvesicles through low-oxygen pre-stimulation, ultrasonic-calcium ion carrier collaborative induction and differential centrifugation.

Inventors

  • ZHANG ZHENGLIANG
  • LI HONGBO

Assignees

  • 安徽科门生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260203

Claims (10)

  1. 1. A method for preparing microvesicles based on stem cells, comprising the steps of: 1) Culturing stem cells in a complete culture medium until the cell fusion degree is 80-90%, performing hypoxia treatment, discarding the culture medium, and cleaning for 2-3 times; 2) Adopting a serum-free basic culture medium to statically culture the stem cells washed in the step 1), and then adding a calcium ion carrier into the serum-free basic culture medium to carry out ultrasonic treatment; 3) Immediately absorbing a serum-free basic culture medium containing calcium ion carriers after the ultrasonic treatment in the step 2), adopting PBS with the mass fraction of bovine serum albumin of 1% to clean cells for 1-3 times, adding a new serum-free basic culture medium for culturing for 24 hours, and respectively collecting and combining cell supernatants in the 6 th, 12 th and 24 th of the culture; 4) The method comprises the steps of first centrifugation to remove cell fragments, second centrifugation to remove dead cells and large cell fragments, third centrifugation to remove organelles, fourth centrifugation to remove large vesicles to obtain a final supernatant; 5) Filtering the final supernatant obtained in the step 4) by using a filter membrane, centrifuging for 70-90 min at 4 ℃ and 100000-120 g, and discarding the supernatant to obtain microvesicle precipitate; 6) Resuspension the microvesicle pellet obtained in step 5), subpackaging, and storing in a refrigerator at-80 ℃.
  2. 2. The preparation method according to claim 1, wherein the stem cells in the step 1) are mesenchymal stem cells, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, amniotic mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells, the environmental conditions for the culture in the step 1) are 37 ℃ and CO 2 % by volume, the environmental conditions for the hypoxia treatment in the step 1) are 37 ℃ and CO 2 % by volume, the O 2 % by volume is 2%, the hypoxia treatment time is 10-14 h, and the sterile PBS is used for the washing in the step 1).
  3. 3. The preparation method according to claim 1, wherein the stationary culture is performed for 0.5-1 h in the step 2), the stationary culture is performed under the environmental conditions of 37 ℃ and CO 2 volume fraction of 5%, and the final concentration of the calcium ionophore in the serum-free basal medium in the step 2) is 1 mu M.
  4. 4. The method according to claim 1, wherein in the step 2), the ultrasonic treatment is performed using a degassed and preheated sterile physiological saline as an ultrasonic coupling agent, the ultrasonic treatment has a frequency of 200kHz, the ultrasonic treatment has a power of 0.5 to 1.5w/cm 2 , the ultrasonic treatment is performed with a pulse wave for 0.5s and is suspended for 0.5s, and the ultrasonic treatment is performed for 2 to 4min.
  5. 5. The method according to claim 1, wherein the environmental conditions of the cultivation in step 3) are 37℃and CO 2 % by volume.
  6. 6. The method according to claim 1, wherein the first centrifugation is performed at a temperature of 4 ℃ for 10 minutes, the second centrifugation is performed at a temperature of 4 ℃ for 2000g for 20 minutes, the third centrifugation is performed at a temperature of 4 ℃ for 10000g for 30 minutes, and the fourth centrifugation is performed at a temperature of 4 ℃ for 20000g for 45 minutes.
  7. 7. The process according to claim 1, wherein the filter used in step 5) is a 0.22 μm filter.
  8. 8. The method of claim 1, wherein the resuspension in step 6) is performed with sterile PBS or sterile physiological saline.
  9. 9. The microvesicles produced by the production process according to any one of claims 1 to 8.
  10. 10. Use of a microvesicle according to claim 9 for the preparation of a product for repairing tissue damage.

Description

Stem cell-based microvesicle and preparation method and application thereof Technical Field The invention belongs to the technical field of biological tissue engineering, and particularly relates to a stem cell-based microvesicle, and a preparation method and application thereof. Background Stem cell therapy has great potential in the fields of tissue repair, regenerative medicine, disease treatment, and the like. However, direct application of stem cells presents many challenges such as immune rejection, tumorigenesis risk, ethical constraints, low in vivo survival rates, and low fixation efficiency. In recent years, research on paracrine mechanisms of stem cells has revealed that one of the key mechanisms of the therapeutic action of stem cells is through secretion of various active factors, and microvesicles (e.g., exosomes, microvesicles, etc.) are critical signaling vectors therein. The nanoscale or microscale membranous vesicles carry bioactive substances such as proteins, nucleic acids (such as mRNA and miRNA), lipids and the like of source cells, can mediate intercellular communication, regulate biological behaviors of receptor cells, and have remarkable effects in the aspects of promoting angiogenesis, inhibiting apoptosis, regulating immune response, stimulating tissue regeneration in situ and the like. At present, the acquisition of stem cell microvesicles mainly depends on the separation and purification from a conditioned medium after in vitro culture of stem cells. The conventional preparation method mostly adopts an ultracentrifugation method, and combines ultrafiltration, size exclusion chromatography, polymer precipitation and other technologies for purification. However, the prior art has a number of significant bottlenecks, severely restricting the clinical transformation and industrialization applications of microvesicles. The stem cells have limited natural secretion of microvesicles under conventional conditions, so that only a very small amount of microvesicles can be harvested from a large amount of culture medium, the production cost is extremely high, and the requirements of large-scale treatment or commercial products on dose consistency and sufficiency are difficult to meet. Most of the existing methods passively collect microvesicles naturally secreted by stem cells, and lack an effective means of actively, directionally pre-orienting stem cells to "promote" their generation of microvesicles. The state of stem cells, the culture microenvironment, specific induction factors, etc. have a decisive influence on the yield and the content composition of microvesicles, but the conventional culture systems do not systematically optimize these factors to achieve "high yield" of microvesicles. Therefore, the preparation method capable of efficiently generating the microvesicles and remarkably improving the yield of the microvesicles is developed, simultaneously the bioactivity and the functional uniformity of the obtained microvesicles are ensured and even enhanced as much as possible, and the method has important significance for promoting the basic research, clinical treatment application and related product development of stem cell microvesicles. The preparation method not only can solve the core pain point with low yield and high cost in the current preparation technology, but also can lay a solid foundation for obtaining the microvesicle treatment product with controllable quality and definite efficacy. Disclosure of Invention In order to solve the technical problems, the invention provides a stem cell-based microvesicle, a preparation method and application thereof, and the preparation method of the microvesicle aims at systematically improving the yield and the bioactivity of the stem cell microvesicle through low-oxygen pre-stimulation, ultrasonic-calcium ion carrier collaborative induction and differential centrifugation refinement collection flow. The invention provides a preparation method of microvesicles based on stem cells, which comprises the following steps of 1) culturing stem cells in a complete culture medium until the cell fusion degree is 80-90%, performing hypoxia treatment, discarding the culture medium, and cleaning for 2-3 times; 2) adopting a serum-free basic culture medium to perform stationary culture on the stem cells after the cleaning in the step 1), then adding a calcium ion carrier into the serum-free basic culture medium to perform ultrasonic treatment, 3) immediately sucking the serum-free basic culture medium containing the calcium ion carrier after the ultrasonic treatment in the step 2), adopting PBS with the mass fraction of bovine serum albumin of 1% to clean the cells for 1-3 times, adding a new serum-free basic culture medium to perform culture for 24 hours, respectively collecting and combining cell supernatants in the 6 th, 12 th and 24 th hours of culture, 4) performing first centrifugation to remove cell fragments, performing second centrifuga