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Methods for complex tissue engineering

A complex tissue and bioengineering technology, applied in biochemical equipment and methods, tissue culture, bone/connective tissue cells, etc., can solve problems such as inability to prepare

Active Publication Date: 2013-03-13
VERSITECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Second, integration with host cartilage is a common problem with most existing strategies, as multiple cylindrical plugs are often used to fill defects
Tissues with bulky matrices, tissues requiring mesenchymal cell types, load-bearing functions, and tissues with irregular tissue junctions, etc. cannot be prepared using this method

Method used

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  • Methods for complex tissue engineering
  • Methods for complex tissue engineering
  • Methods for complex tissue engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Bone marrow aspiration and separation of rabbit bone marrow mesenchymal stem cells (rMSC)

[0075] Three-month-old New Zealand White rabbits weighing an average of 3.5 kg were anesthetized by intramuscular injection of a mixture of 10% ketamine hydrochloride (0.35 ml / kg) and 2% xylazine (0.25 ml / kg). About 5 ml of bone marrow was aspirated from the tibia. After Ficoll-Hypague gradient separation, monocytes at the interface were collected and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics. The medium was refreshed 10 days after inoculation and refilled every 2 days thereafter. Visible colonies of adherent cells were found approximately 5-7 days after initial plating. After reaching confluence (approximately 12-14 days after initial plating), cells were detached by 0.25% trypsin / EDTA for subculture.

[0076] Alternatively, rMSCs can be purchased from Beijing YiKeLiHao Biotechnology Co., Ltd.

Embodiment 2

[0077] Embodiment 2: the culture of rMSC

[0078] In Dulbecco's modified Eagle medium (DMEM-HG) with high glucose, 10% fetal bovine serum (FBS), 100U / ml penicillin, 100mg / ml streptomycin, 1.875mg / ml sodium bicarbonate (NaHCO 3 ), 0.02M HEPES and 0.29mg / ml L-glutamine to culture rMSC in the complete medium. The final pH of the medium was adjusted to 7.4 with 1 N sodium hydroxide (NaOH). Live cells in culture were separated from dead cells after 24 hours by adhesion selection, ie, cells were cultured for 24 hours and adherent cells were separated from dead cells in culture. Maintain cells in complete medium and refill every 3 days. Subconfluence rMSCs were detached by 0.25% trypsin / EDTA. Cells from passage 2-3 were used in the subsequent microencapsulation step.

Embodiment 3

[0079] Example 3: Fabrication of Naïve Subunit-Collagen-rMSC Microspheres

[0080] Ice-cold rat tail type I collagen (Becton Dickenson) was neutralized with 1 N NaOH and further diluted with complete medium to a final concentration of 2 mg / ml. Aliquots of rMSCs from P2-P3 in complete medium were quickly mixed with neutralized collagen solution in an ice bath, resulting in a cell-matrix mixture with a final cell density of 1250 cells per 2.5 μl drop. Droplets were dispensed into 35mm diameter Petri dishes (Sterlin) with UV irradiated parafilm on the bottom layer. With 5% CO 2 After incubation for 1 hour at 37°C in a humidified atmosphere, the droplets gelled to form solid rMSC-collagen microspheres, which were then gently rinsed into Petri dishes using complete medium and incubated for 3 hours prior to the differentiation step. sky.

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Abstract

A simple, highly flexible and scalable platform for making functional complex tissue with heterogeneity and irregularity is provided. The method includes combining undifferentiated cells, such as pluripotent or multipotent stem cells, with a biomaterial to make multiple undifferentiated or naive subunits, exposing the undifferentiated or naive subunits to different cell culture environments for induction of differentiation towards different lineages as required by said complex tissue, and combining the then functional subunits solely or with the undifferentiated subunits. The differentiated subunits thus combined can be cultured under biological, chemical, and / or physical culture conditions suitable to fine-tune the structural and functional properties of the bioengineered complex tissue to form a bioengineered tissue graft that mimics the structural and functional characteristics of native complex tissue. Said bioengineered tissue graft can be used to replace dysfunctional tissue.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 61 / 354,869 filed June 15, 2010, which is hereby incorporated by reference in its entirety. field of invention [0003] The present invention relates generally to bioengineered tissues. In particular, it relates to methods of producing tissue grafts having more than one tissue component. Background of the invention [0004] Traumatic, genetic or surgical causes can cause tissue dysfunction. With mild injuries, some tissues are able to regenerate themselves, while other tissues, especially those of a hypovascular nature, barely regenerate. Numerous attempts have been made to provide treatment options for tissue dysfunction, including growth factor therapy, cell therapy, and gene therapy. However, these attempts are only useful if the degree of tissue dysfunction is not great. Alternative therapy through surgical approaches is necessary when tissue ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/40A61L27/38C12N5/07C12N5/0735C12N5/0775C12N5/0789A61K35/28A61K35/51A61K35/545
CPCC12N2502/1317C12N5/0012A61L27/3834A61K35/51C12N2502/1358C12N2527/00C12N5/0654C12N5/0697C12N2502/1311A61K2035/128A61L27/3891C12N5/0655A61K35/545C12N2533/54A61K35/28A61P43/00
Inventor 陈佩郑晓华戚子杰张文智陆瓞骥
Owner VERSITECH LTD
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