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Human residual ear cartilage stem cells and method for constructing tissue engineered cartilage

A technique for chondrocyte and tissue engineering, applied in the fields of medicine and biomedical engineering

Active Publication Date: 2016-01-20
SHANGHAI TISSUE ENG LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the source of seed cells has always been a bottleneck restricting the clinical transformation of this technology
Then, is it possible to use the autologous residual ear chondrocytes of patients with microtia to construct auricular cartilage for ear reconstruction? To realize this dream, we must first solve three key problems: 1. The number of cells: only 1-3×10 cells can be isolated from the residual ear cartilage of a patient. 6 cells, can these cells be expanded to the number of cells required for the construction of normal-sized human ear shape cartilage (1.5-3×10 8 cells)? 2. Cell function question: Do the expanded remnant ear chondrocytes still have good cartilage formation ability? 3. Construction technical question: How to use these cells to construct cartilage with precise shape of human auricle in vitro?

Method used

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  • Human residual ear cartilage stem cells and method for constructing tissue engineered cartilage
  • Human residual ear cartilage stem cells and method for constructing tissue engineered cartilage
  • Human residual ear cartilage stem cells and method for constructing tissue engineered cartilage

Examples

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preparation example Construction

[0063] The preparation method of the tissue-engineered cartilage of the present invention is simple and convenient. A certain amount of the human remnant ear chondrocytes is mixed with a pharmaceutically acceptable biodegradable material, and then the cartilage is induced in vitro. The method comprises the steps of:

[0064] In the first step, the cells derived from human residual ear cartilage are cultured and amplified, and subcultured to 1-8 generations;

[0065] In the second step, the above-mentioned subcultured and expanded cells are centrifuged to form pellets or high-density seeding to form cell membranes; or these cells are mixed with solid medically acceptable biodegradable materials to form cell-biomaterial composites thing;

[0066] In the third step, the agglomerates, membrane sheets or complexes are placed in chondrogenic induction medium for in vitro culture.

[0067] The method of planar culture expansion in the first step is, for example but not limited to, ...

Embodiment 1

[0103] Acquisition and culture of cartilage stem cells from human remnant ear

[0104] The cells are taken from the autologous residual ear cartilage tissue of patients with microtia. The donors are 8-30 years old, healthy, and free from malignant tumors, infectious diseases and autoimmune diseases.

[0105] (1) Material collection: Under aseptic conditions in the operating room, after satisfactory anesthesia, drape was routinely sterilized, and the patient's residual ear tissue was cut out, placed in a 50mL sterile centrifuge tube, soaked in 40mL sterile saline for storage and transport.

[0106] (2) Separation of residual ear chondrocytes: Under sterile conditions in an ultra-clean bench, completely remove the connective tissue and perichondrium around the residual ear cartilage, and chop the cartilage to 1-2mm 3 Small pieces were soaked in 0.25% chloramphenicol solution for 30-40min, rinsed with PBS, digested with 0.25% trypsin + 0.02% EDTA (Amresco, USA) in a constant temp...

Embodiment 2

[0112] Preparation of pellets from remnant ear cartilage stem cells

[0113] (1) Cell collection: when the monolayer cultured human residual ear chondrocytes reached 90% confluence, they were digested with 0.25% trypsin+0.02% EDTA. After the cells were collected, they were counted with a cell counter. Trypan blue staining showed that the number of viable cells was more than 90%. The cells were centrifuged at 1800 rpm for 8 min to precipitate the cells. The supernatant was discarded, and the cells were dispersed by shaking.

[0114] (2) Preparation of pellets: After collecting the cells, resuspend them in ordinary culture medium to prepare 0.4×10 pellets. 6 / ml of cell suspension. Take 1ml of the suspension in a 15ml centrifuge tube and centrifuge at 1000rpm for 5min to make a cell pellet. Slightly loosen the bottle cap and place at 37°C, 5% CO 2 , cultured in an incubator with saturated humidity, and then changed the medium every other day (ordinary culture medium or chondr...

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Abstract

The invention discloses a tissue engineered cartilage graft. The tissue engineered cartilage graft contains human residual ear chondrocytes grown in a three-dimensional structure; the human residual ear chondrocytes are the first, second, third, 4, 5, 6, 7, or 8 passages. The invention also discloses the application of human remnant ear chondrocytes in constructing cartilage grafts in vitro.

Description

technical field [0001] The invention relates to the fields of medicine and biomedical engineering, and more particularly relates to tissue-engineered cartilage constructed by human residual ear cartilage stem cells, a preparation method and application thereof. Background technique [0002] Microtia (microtia) is a congenital disease with a relatively high incidence (about 1 / 3000), which is characterized by the loss of auricular landmarks, and the ear area is replaced by contracted and deformed cartilage masses (residual ear cartilage). At present, the clinical treatment of microtia is mainly to take the patient's autologous costal cartilage into the shape of the auricle and then implant it under the skin of the ear area. The main disadvantage of this method is that it causes a lot of trauma to the normal costal cartilage, and it is easy to cause complications at the donor site such as pneumothorax, thorax collapse, deformity and scar. [0003] The rise and rapid developmen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/38
Inventor 周广东刘豫曹谊林刘伟
Owner SHANGHAI TISSUE ENG LIFE SCI
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