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CN-122012382-A - Periosteum-like organ culture method and application thereof

CN122012382ACN 122012382 ACN122012382 ACN 122012382ACN-122012382-A

Abstract

The invention relates to a periosteum organ culture method and application thereof, belonging to the technical field of biomedicine. The invention provides a periosteum organoid culture method which comprises the following steps of (1) digesting periosteum tissues to obtain cell resuspension, (2) centrifuging the cell resuspension to obtain periosteum cells, mixing the periosteum cells with basement membrane matrixes to obtain periosteum cell resuspension, and (3) mixing the periosteum cell resuspension with a ROCK inhibitor, and adding the mixture into an organoid culture medium to culture to obtain a periosteum organoid. The invention constructs the first periosteum organoid which can be amplified in vitro for a long time, can effectively inhibit the aging of HSC in the in vitro culture process, verifies the repairing effect of the periosteum organoid on bone defect by using femur and skull defect models, and reveals the potential application of the periosteum organoid to bone tissue regeneration.

Inventors

  • ZHOU YONGSHENG
  • ZHANG PING
  • QING JIA
  • Gu lanxin
  • FU LIPENG

Assignees

  • 北京大学口腔医学院

Dates

Publication Date
20260512
Application Date
20260212

Claims (10)

  1. 1. A method for culturing periosteal organoids, comprising the steps of: (1) Mixing periosteum tissue with collagenase digestive juice, digesting for 25-35 min, filtering the supernatant to obtain digestive juice, mixing the digestive juice with primary extraction buffer solution to obtain mixed solution, centrifuging for 5-15 min, and mixing the precipitate with the primary extraction buffer solution to obtain cell heavy suspension; (2) Purifying the cell re-suspension, centrifuging for 1-10 min to obtain periosteum cells, and mixing the periosteum cells with basement membrane matrix to obtain periosteum cell re-suspension; (3) Mixing periosteum cell resuspension and a ROCK inhibitor to obtain a culture solution, mixing the culture solution with an organoid culture medium, and culturing for 7-14 days to obtain the periosteum organoid.
  2. 2. The culture method according to claim 1, wherein the periosteum tissue and collagenase digestion solution are mixed in the volume ratio of 1.5:6-8 in the step (1); the collagenase digestion solution takes water as a solvent and comprises collagenase P with the final concentration of 0.5-1.5 mg/mL and dispase II with the final concentration of 0.5-1.5 mg/mL.
  3. 3. The method according to claim 1, wherein the digestion in step (1) is carried out at a temperature of 35 to 39℃and a rotation speed of 750 to 850rpm.
  4. 4. The culture method according to claim 1, wherein the volume ratio of the digestion solution to the primary extraction buffer solution in the step (1) is 1-2:1-2; the primary extraction buffer solution in the step (1) takes PBS buffer solution as a solvent and comprises 3-5 vt% of fetal bovine serum and 1-3 vt% of penicillin-streptomycin.
  5. 5. The culture method according to claim 1, wherein the centrifugal force of the centrifugation in the step (1) is 200 to 300g and the temperature is 3 to 5 ℃; the cell concentration of the cell resuspension is 5-10 multiplied by 10 6 /mL.
  6. 6. The culture method according to claim 1, wherein the centrifugal force of the centrifugation in the step (2) is 200 to 300g and the temperature is 3 to 5 ℃; The cell concentration of the periosteum cell resuspension is 4-6 multiplied by 10 4 /mL.
  7. 7. The culture method according to claim 1, wherein the final concentration of ROCK inhibitor in the culture solution in the step (3) is 5 to 15 μm; the volume ratio of the culture solution to the organoid culture medium is 4:70-300.
  8. 8. The culture method according to claim 1, wherein the organoid medium in step (3) is based on an alpha-MEM medium comprising the following concentrations of components: 1-3 vt% of penicillin-streptomycin, 5-15 mM of 4-hydroxyethyl piperazine ethane sulfonic acid, 1-3 mM of cell culture additive, 0.5-1.5 vt% of B-27 additive, 1-3 vt% of N-2 additive, 15-25 ng/mL of alkaline fibroblast growth factor, 15-25 ng/mL of Oncoinhibin M, 15-25 ng/mL of epidermal growth factor, 35-45 ng/mL of insulin-like growth factor and 10-20 vt% of chick embryo extract.
  9. 9. The periosteal organoid cultured by the culture method according to any one of claims 1 to 8.
  10. 10. Use of the periosteal organoid of claim 9 for the manufacture of a product for the treatment of bone defect disorders.

Description

Periosteum-like organ culture method and application thereof Technical Field The invention relates to the technical field of biomedicine, in particular to a periosteum organoid culture method and application thereof. Background Bone injury is mechanical injury caused by external impact, falling, torsion and the like, or is collectively called as bone tissue structural integrity damage caused by pathological factors such as osteoporosis, bone tumor, infection and the like, a plurality of urgent problems still exist in the current bone injury treatment, firstly, the clinical problems related to bone healing are solved, partial injury is caused by severe periosteum blood transport damage and unstable fixation, or patients are combined with basic diseases such as diabetes, osteoporosis and the like, delayed bone healing, non-healing or malformation healing are easy to occur, and partial cases need secondary operation intervention, so that pain and medical burden of the patients are increased; secondly, the control difficulty of operation complications is that the traditional open operation is large in wound, wound infection is easy to cause, internal fixtures are loosened and broken, the minimally invasive technology reduces the wound, but the requirement on operation accuracy is high, the operation is difficult to popularize in basic medical institutions, meanwhile, functional disorders such as joint stiffness and muscle atrophy after operation are common, and the timing and strength of rehabilitation training are not uniform and have individual standards; in addition, the bone repair material has the defects of clinical application, namely gold standard, but has the problems of insufficient donor and pain in the bone taking area, the problems of poor biocompatibility, insufficient bone conduction and bone induction capacity of allogeneic bone and artificial bone materials are encountered, the autologous bone can not be completely replaced, meanwhile, the treatment scheme of old bone injury patients is limited, the patients mostly incorporate multiple basic diseases, the operation tolerance is poor, the conservative treatment is easy to cause pneumonia due to long-term bedridden patients, and the like, complications such as pressure sores, and the like are difficult to balance treatment effect and safety risk in clinic, and the situation that partial individuation scheme is not formulated enough still exists in the current clinic treatment, so that the problems of over treatment or under treatment easily occur, and the overall treatment effect of bone injury is affected. Based on this, the present invention has been proposed. Disclosure of Invention The invention aims to provide a periosteum organ culture method and application thereof, which are used for solving the problems of bone injury treatment in the prior art. In order to achieve the above object, the present invention provides the following technical solutions: the invention provides a periosteum organoid culture method, which comprises the following steps: (1) Mixing periosteum tissue with collagenase digestive juice, digesting for 25-35 min, filtering the supernatant to obtain digestive juice, mixing the digestive juice with primary extraction buffer solution to obtain mixed solution, centrifuging for 5-15 min, and mixing the precipitate with the primary extraction buffer solution to obtain cell heavy suspension; (2) Purifying the cell re-suspension, centrifuging for 1-10 min to obtain periosteum cells, and mixing the periosteum cells with basement membrane matrix to obtain periosteum cell re-suspension; (3) Mixing periosteum cell resuspension and a ROCK inhibitor to obtain a culture solution, mixing the culture solution with an organoid culture medium, and culturing for 7-14 days to obtain the periosteum organoid. Preferably, in the step (1), the periosteum tissue and the collagenase digestion solution are mixed in a volume ratio of 1.5:6-8; the collagenase digestion solution takes water as a solvent and comprises collagenase P with the final concentration of 0.5-1.5 mg/mL and dispase II with the final concentration of 0.5-1.5 mg/mL. Preferably, the digestion temperature in step (1) is 35-39 ℃ and the rotation speed is 750-850 rpm. Preferably, in the step (1), the volume ratio of the digestion solution to the primary extraction buffer solution is 1-2:1-2; the primary extraction buffer solution in the step (1) takes PBS buffer solution as a solvent and comprises 3-5 vt% of fetal bovine serum and 1-3 vt% of penicillin-streptomycin. Preferably, the centrifugal force of the centrifugation in the step (1) is 200-300 g, and the temperature is 3-5 ℃; the cell concentration of the cell resuspension is 5-10 multiplied by 10 6/mL. Preferably, the centrifugal force of the centrifugation in the step (2) is 200-300 g, and the temperature is 3-5 ℃; The cell concentration of the periosteum cell resuspension is 4-6 multiplied by 10 4/mL. Preferably, the final conc