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CN-122012385-A - Culture method of high-concentration composite exosomes

CN122012385ACN 122012385 ACN122012385 ACN 122012385ACN-122012385-A

Abstract

The invention discloses a culture method of a high-concentration compound exosome, aiming at the continuous high-yield culture problem of exosome culture, the culture scheme of the exosome is improved, and through cross passage and starvation time sequence control, umbilical cord mesenchymal stem cells can synchronously realize proliferation of cells and collection of the exosome in a short time, and the differential centrifugal purification process arranged in the subsequent step of the invention can also remove fragments with different sizes in supernatant through multistage centrifugal rates with different gradients, thereby realizing that the purity of the exosome is ensured while the yield of the exosome is increased.

Inventors

  • LIN YIMAN
  • LIN LUSHENG
  • CHEN ZHESHENG

Assignees

  • 深圳市和创众盈生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260210

Claims (10)

  1. 1. The culture method of the high-concentration compound exosome is characterized by comprising the following steps of: S1, cell culture; s11, inoculating and culturing cells; thawing, resuscitating and primarily culturing the P3-generation umbilical cord mesenchymal stem cells; s12, cell subculturing; After the primary culture is finished, detecting the growth condition of the cells, and when the cells are grown to 80-90% in a fusion way, carrying out subculture to the third generation to obtain a third generation T-225 cell culture bottle; S2, exosome separation and purification; s21, selecting a third-generation T-225 cell culture bottle accounting for 60-70% of the total number, replacing the third-generation T-225 cell culture bottle with a serum-free culture medium, and performing starvation culture on the third-generation T-225 cell culture bottle for 48-72 hours to obtain a third-generation starvation culture T-225 cell culture bottle; Processing the rest third-generation T-225 cell culture flasks according to the step S12, and further carrying out subculture until the number of the third-generation T-225 cell culture flasks is the same as that of the third-generation T-225 cell culture flasks in the step S12, and continuing culturing for 48-72 h to obtain a fourth-generation T-225 cell culture flask; S22, collecting supernatant in the third-generation starvation culture T-225 cell culture flask in the step S21, and after centrifugal separation, uniformly mixing the supernatant to obtain a first exosome supernatant; Selecting a fourth-generation T-225 cell culture bottle with 60-70% of the culture bottles, replacing the culture bottle with a serum-free culture medium, adding a first batch of exosome supernatant into the culture bottle, and performing starvation culture for 48-72 hours to obtain a fourth-generation starvation culture T-225 cell culture bottle; S12, processing the rest fourth-generation T-225 cell culture flasks, and performing further subculture until the number of the fourth-generation T-225 cell culture flasks is the same as that of the fourth-generation starvation culture T-225 cell culture flasks, and continuing culturing for 48-72 hours to obtain a fifth-generation T-225 cell culture flask; S23, collecting supernatant in the fourth-generation starvation culture T-225 cell culture flask in the step S22, and after centrifugal separation, uniformly mixing the supernatant to obtain supernatant of a second exosome; replacing a fifth-generation T-225 cell culture bottle with a serum-free culture medium, adding a second batch of exosome supernatant into the culture bottle, starving the culture bottle for 48-72 hours, collecting the supernatant in the cell culture bottle, and centrifugally separating to obtain a high-concentration composite exosome supernatant; S3, performing differential centrifugal purification treatment on the high-concentration composite exosome supernatant obtained in the step S2 to obtain the high-concentration composite exosome.
  2. 2. The method for culturing high-concentration complex exosomes according to claim 1, wherein in step S11, thawing, resuscitating and primary culturing are performed, specifically comprising the steps of: Taking P3 generation umbilical cord mesenchymal stem cells, placing the cells in a constant-temperature water bath kettle for melting in a water bath at 37 ℃, pouring the cells into a 50 mL-specification centrifuge tube filled with 30mL of DPBS buffer solution, sealing a mouth of the centrifuge tube, repeatedly reversing and shaking the cells uniformly, and centrifuging the cells; And after centrifugation, taking supernatant to perform sterile detection, pouring out redundant supernatant in a centrifuge tube after no pollution is detected, taking centrifuged cells and culture medium to be evenly resuspended, equally dividing and inoculating the cells and the culture medium into 2 bottles of T-225 cell culture flasks, adding the culture medium to 40mL, and placing the T-225 cell culture flasks into a cell culture incubator with constant temperature of 37 ℃ and concentration of 5% CO 2 for culturing for 48-72 h to complete primary culture.
  3. 3. The method for culturing high-concentration complex exosomes according to claim 1, wherein in the steps S1 and S2, the specific steps are as follows: Pouring the original culture medium in a T-225 cell culture bottle, adding DPBS buffer solution into the culture bottle according to the proportion of 8-12 mL/T-225 cell culture bottle to clean cells, pouring the DPBS buffer solution, adding recombinant pancreatin into the culture bottle according to the proportion of 4-6 mL/T-225 cell culture bottle to digest cells for 2-3 min, adding the DPBS buffer solution into the culture bottle according to the proportion of 8-12 mL/T-225 cell culture bottle to terminate digestion, adding the cell suspension into a centrifuge tube, centrifuging at 1200-630 rpm for 4-6 min, re-suspending cells by using the culture medium, adding the culture medium into a new T-225 cell culture bottle, and supplementing the culture medium to the volume of 40mL/T-225 cell culture bottle.
  4. 4. The method for culturing high-concentration complex exosomes according to claim 1, wherein in the steps S1 and S2, the passaging ratio is 1:2-4.
  5. 5. The method of claim 1, wherein in step S1, the medium formulation is a serum-free medium containing 4-6% UltraGRO-Advanced alpha-MEN during primary culture and subculture.
  6. 6. The method of claim 1, wherein in step S2, the culture medium formulation is a serum-free medium containing 4-6% UltraGRO-Advanced alpha-MEN.
  7. 7. The method of claim 1, wherein in step S2, the serum-free medium comprises 0 to 0.1. Mu. Mol/L of labyrine A and 0 to 5. Mu. Mol/L of Rho kinase inhibitor.
  8. 8. The method of claim 1, wherein in step S2, the first and second supernatants of the high-concentration complex exosomes are prepared by centrifugation as follows: Adding the supernatant into a centrifuge tube, cooling to 4 ℃, centrifuging 300-400 g for 8-12 min, centrifuging 1800-2500 g for 20min, taking the supernatant in the centrifuge tube, passing through a cell filter screen of 100-200 mu m, collecting filtrate, collecting, sub-packaging and centrifuging.
  9. 9. The method for culturing high-concentration complex exosomes according to claim 1, wherein in step S3, the specific steps are as follows: Adding the high-concentration composite exosome supernatant into a centrifuge tube, cooling to 4 ℃, centrifuging for 10-12 min at 300-350 g, centrifuging for 25-40 min at 9000-10000 g, centrifuging for 70-90 min at 90000-100000 g again, taking out the centrifuge tube, sucking the supernatant after centrifugation, reserving a 0.5cm liquid layer to avoid exosome loss, and completing differential centrifugation purification.
  10. 10. A high-concentration complex exosome cultured by the culture method according to any one of claims 1 to 9.

Description

Culture method of high-concentration composite exosomes Technical Field The invention relates to the technical field of exosomes, in particular to a culture method of a high-concentration compound exosome. Background Exosomes (exosomes) are taken as micro-membrane vesicles widely existing in cell secretion products, the diameter of the micro-membrane vesicles is between 30 and 150nm, lipid bilayer membranes are taken as a structural basis, and the micro-membrane vesicles contain multiple functional molecules such as cytokines, growth factors, lipid substances, coded mRNA and the like, so that the micro-membrane vesicles can effectively mediate information exchange among cells, remodel local microenvironment of tissues and play an indispensable key regulation role in various physiological and pathological processes such as immunoregulation, tissue injury repair and the like, but the conventional culture scheme of the exosomes often has the problems of low yield, strong heterogeneity and excessive purification loss, the prior art adopts single starvation culture without cross passage, cell proliferation and exosomes secretion are asynchronous, and cell debris cannot be effectively removed by adopting single centrifugation rate in purification, so that the problem of large-scale culture of the exosomes exists, and continuous production cannot be realized. Disclosure of Invention The invention aims to provide a culture method of a high-concentration compound exosome, which aims to solve the problems in the prior art. In order to achieve the aim, the invention provides the following technical scheme that the culture method of the high-concentration compound exosome comprises the following steps: S1, cell culture; s11, inoculating and culturing cells; thawing, resuscitating and primarily culturing the P3-generation umbilical cord mesenchymal stem cells; s12, cell subculturing; After the primary culture is finished, detecting the growth condition of the cells, and when the cells are grown to 80-90% in a fusion way, carrying out subculture to the third generation to obtain a third generation T-225 cell culture bottle; S2, exosome separation and purification; s21, selecting a third-generation T-225 cell culture bottle accounting for 60-70% of the total number, replacing the third-generation T-225 cell culture bottle with a serum-free culture medium, and performing starvation culture on the third-generation T-225 cell culture bottle for 48-72 hours to obtain a third-generation starvation culture T-225 cell culture bottle; Processing the rest third-generation T-225 cell culture flasks according to the step S12, and further carrying out subculture until the number of the third-generation T-225 cell culture flasks is the same as that of the third-generation T-225 cell culture flasks in the step S12, and continuing culturing for 48-72 h to obtain a fourth-generation T-225 cell culture flask; S22, collecting supernatant in the third-generation starvation culture T-225 cell culture flask in the step S21, and after centrifugal separation, uniformly mixing the supernatant to obtain a first exosome supernatant; Selecting a fourth-generation T-225 cell culture bottle with 60-70% of the culture bottles, replacing the culture bottle with a serum-free culture medium, adding a first batch of exosome supernatant into the culture bottle, and performing starvation culture for 48-72 hours to obtain a fourth-generation starvation culture T-225 cell culture bottle; S12, processing the rest fourth-generation T-225 cell culture flasks, and performing further subculture until the number of the fourth-generation T-225 cell culture flasks is the same as that of the fourth-generation starvation culture T-225 cell culture flasks, and continuing culturing for 48-72 hours to obtain a fifth-generation T-225 cell culture flask; S23, collecting supernatant in the fourth-generation starvation culture T-225 cell culture flask in the step S22, and after centrifugal separation, uniformly mixing the supernatant to obtain supernatant of a second exosome; replacing a fifth-generation T-225 cell culture bottle with a serum-free culture medium, adding a second batch of exosome supernatant into the culture bottle, starving the culture bottle for 48-72 hours, collecting the supernatant in the cell culture bottle, and centrifugally separating to obtain a high-concentration composite exosome supernatant; S3, performing differential centrifugal purification treatment on the high-concentration composite exosome supernatant obtained in the step S2 to obtain the high-concentration composite exosome. Further, in step S11, the thawing recovery and the primary culture specifically include the following steps: Taking P3 generation umbilical cord mesenchymal stem cells, placing the cells in a constant-temperature water bath kettle for melting in a water bath at 37 ℃, pouring the cells into a 50 mL-specification centrifuge tube filled with 30mL of DPBS buffer solution, s