Application of umbilical cord Huatong jelly mesenchymal stem cells in the preparation of cell transplantation materials

Abstract

The invention relates to the application of umbilical cord Huatong jelly mesenchymal stem cells in the preparation of medical biological materials. This medical biomaterial can be used for human intervertebral disc cell transplantation, and can also be used as seed cells for intervertebral disc tissue engineering. It uses fresh human umbilical cord as material to prepare Huatong jelly mesenchymal stem cells, and uses the intervertebral disc specimen removed during intervertebral discectomy as material to isolate and culture normal nucleus pulposus cells; then apply non-contact or contact cell co-culture method to induce Differentiation of umbilical cord Huatong jelly mesenchymal stem cells.

Description

technical field

[0001] The invention relates to the application of biological materials in the preparation of medical materials, in particular to the application of mesenchymal stem cells prepared from human umbilical cord Huatong jelly in the preparation of cell transplantation materials and tissue engineering seed cell materials. The medical materials prepared by the method of the invention, including cell transplantation materials and seed cell materials, can be used for the treatment of intervertebral disc degenerative diseases. Background technique

[0002] Degenerative disc disease (DDD) is a common and frequently-occurring disease, and the resulting low back pain often seriously affects the work and life of patients. The intervertebral disc is composed of gelatinous nucleus pulposus tissue surrounded by fibrous rings and upper and lower cartilage endplates. The gelatinous nucleus pulposus tissue contains a large amount of proteoglycan and type II collagen. Proteoglyca...

Examples

Embodiment 1

[0011] Example 1 Isolation and culture of Huatong jelly mesenchymal stem cells

[0012] About 30 cm of fresh neonatal umbilical cord was taken as material, stored aseptically in D-Hanks solution, and stored at 2-6°C. The umbilical cord material is preferably full-term healthy newborn umbilical cord, which can ensure that the umbilical cord develops well; moreover, the umbilical cord of caesarean section newborn is preferred, which has a lower infection rate than vaginal birth.

[0013] Wash the residual blood of the umbilical cord with balanced salt solution in an ultra-clean workbench, cut the umbilical cord into 4cm segments, cut the adventitia of the umbilical cord longitudinally for each segment, remove 2 umbilical arteries and 1 umbilical vein freely, and take the blood vessels The Huatong glue tissue between the blood vessels and the adventitia, cut into about 1mm 3 After washing with balanced salt solution, immerse in collagenase solution with a concentration of 0.2%, ...

Embodiment 2

[0014] Example 2 Isolation and culture of normal nucleus pulposus cells

[0015] Intervertebral discectomy is a common surgical treatment method for patients with intervertebral disc herniation for a long time. The surgical process is to remove or partially remove the displaced intervertebral disc. The present invention collects fresh intervertebral disc specimens removed by operation, immediately separates the nucleus pulposus tissue in an ultra-clean workbench, washes it twice with PBS, and removes residual blood. Mince the nucleus pulposus tissue about 1.0mm 3 Put it into a 5ml centrifuge tube, add 0.2% type II collagenase solution prepared with DMEM / F12 serum-free medium, digest at 37°C for 5 hours, and filter tissue fragments through a sterile stainless steel mesh (200mm). The cell suspension was centrifuged at 1000r / min for 5 minutes, the supernatant was discarded, and the cells were collected, added to DMEM / F12 medium containing 10% fetal bovine serum, and cultured at ...

Embodiment 3

[0016] Example 3 Non-contact cell co-culture method to induce differentiation of umbilical cord Huatong jelly mesenchymal stem cells

[0017] Non-contact co-cultivation is carried out with a Transwell six-well plate with an insert layer. The insert layer has a high-density pore size with a pore size of 0.4 μm, and cytokines can pass through it. The material is polyethylene terephthalate, and the cells can For adherent growth, the insert layer was inoculated with nucleus pulposus cells, and the lower layer of the six-well plate was inoculated with Huatong jelly mesenchymal stem cells. 4 , nucleus pulposus cells are 6×10 4 . After 7 days of culture, Huatong jelly mesenchymal stem cells were collected.

technical field

[0001] The invention relates to the application of biological materials in the preparation of medical materials, in particular to the application of mesenchymal stem cells prepared from human umbilical cord Huatong jelly in the preparation of cell transplantation materials and tissue engineering seed cell materials. The medical materials prepared by the method of the invention, including cell transplantation materials and seed cell materials, can be used for the treatment of intervertebral disc degenerative diseases. Background technique

[0002] Degenerative disc disease (DDD) is a common and frequently-occurring disease, and the resulting low back pain often seriously affects the work and life of patients. The intervertebral disc is composed of gelatinous nucleus pulposus tissue surrounded by fibrous rings and upper and lower cartilage endplates. The gelatinous nucleus pulposus tissue contains a large amount of proteoglycan and type II collagen. Proteoglyca...

Examples

Embodiment 1

[0011] Example 1 Isolation and culture of Huatong jelly mesenchymal stem cells

[0012] About 30 cm of fresh neonatal umbilical cord was taken as material, stored aseptically in D-Hanks solution, and stored at 2-6°C. The umbilical cord material is preferably full-term healthy newborn umbilical cord, which can ensure that the umbilical cord develops well; moreover, the umbilical cord of caesarean section newborn is preferred, which has a lower infection rate than vaginal birth.

[0013] Wash the residual blood of the umbilical cord with balanced salt solution in an ultra-clean workbench, cut the umbilical cord into 4cm segments, cut the adventitia of the umbilical cord longitudinally for each segment, remove 2 umbilical arteries and 1 umbilical vein freely, and take the blood vessels The Huatong glue tissue between the blood vessels and the adventitia, cut into about 1mm 3 After washing with balanced salt solution, immerse in collagenase solution with a concentration of 0.2%, ...

Embodiment 2

[0014] Example 2 Isolation and culture of normal nucleus pulposus cells

[0015] Intervertebral discectomy is a common surgical treatment method for patients with intervertebral disc herniation for a long time. The surgical process is to remove or partially remove the displaced intervertebral disc. The present invention collects fresh intervertebral disc specimens removed by operation, immediately separates the nucleus pulposus tissue in an ultra-clean workbench, washes it twice with PBS, and removes residual blood. Mince the nucleus pulposus tissue about 1.0mm 3 Put it into a 5ml centrifuge tube, add 0.2% type II collagenase solution prepared with DMEM / F12 serum-free medium, digest at 37°C for 5 hours, and filter tissue fragments through a sterile stainless steel mesh (200mm). The cell suspension was centrifuged at 1000r / min for 5 minutes, the supernatant was discarded, and the cells were collected, added to DMEM / F12 medium containing 10% fetal bovine serum, and cultured at ...

Embodiment 3

[0016] Example 3 Non-contact cell co-culture method to induce differentiation of umbilical cord Huatong jelly mesenchymal stem cells

[0017] Non-contact co-cultivation is carried out with a Transwell six-well plate with an insert layer. The insert layer has a high-density pore size with a pore size of 0.4 μm, and cytokines can pass through it. The material is polyethylene terephthalate, and the cells can For adherent growth, the insert layer was inoculated with nucleus pulposus cells, and the lower layer of the six-well plate was inoculated with Huatong jelly mesenchymal stem cells. 4 , nucleus pulposus cells are 6×10 4 . After 7 days of culture, Huatong jelly mesenchymal stem cells were collected.