Method for manufacture and cryopreservation of cartilage microtissues.

IF 6.7 1区 工程技术 Q1 CELL & TISSUE ENGINEERING
Md Shafiullah Shajib, Kathryn Futrega, Rose Ann G Franco, Eamonn McKenna, Bianca Guillesser, Travis J Klein, Ross W Crawford, Michael R Doran
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Abstract

The financial viability of a cell and tissue-engineered therapy may depend on the compatibility of the therapy with mass production and cryopreservation. Herein, we developed a method for the mass production and cryopreservation of 3D cartilage microtissues. Cartilage microtissues were assembled from either 5000 human bone marrow-derived stromal cells (BMSC) or 5000 human articular chondrocytes (ACh) each using a customized microwell platform (the Microwell-mesh). Microtissues rapidly accumulate homogenous cartilage-like extracellular matrix (ECM), making them potentially useful building blocks for cartilage defect repair. Cartilage microtissues were cultured for 5 or 10 days and then cryopreserved in 90% serum plus 10% dimethylsulfoxide (DMSO) or commercial serum-free cryopreservation media. Cell viability was maximized during thawing by incremental dilution of serum to reduce oncotic shock, followed by washing and further culture in serum-free medium. When assessed with live/dead viability dyes, thawed microtissues demonstrated high viability but reduced immediate metabolic activity relative to unfrozen control microtissues. To further assess the functionality of the freeze-thawed microtissues, their capacity to amalgamate into a continuous tissue was assess over a 14 day culture. The amalgamation of microtissues cultured for 5 days was superior to those that had been cultured for 10 days. Critically, the capacity of cryopreserved microtissues to amalgamate into a continuous tissue in a subsequent 14-day culture was not compromised, suggesting that cryopreserved microtissues could amalgamate within a cartilage defect site. The quality ECM was superior when amalgamation was performed in a 2% O2 atmosphere than a 20% O2 atmosphere, suggesting that this process may benefit from the limited oxygen microenvironment within a joint. In summary, cryopreservation of cartilage microtissues is a viable option, and this manipulation can be performed without compromising tissue function.

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软骨显微组织的制备和低温保存方法。
细胞和组织工程疗法的经济可行性可能取决于该疗法与大规模生产和冷冻保存的相容性。在此,我们开发了一种大规模生产和冷冻保存三维软骨显微组织的方法。软骨微组织由5000个人骨髓来源的基质细胞(BMSC)或5000个人关节软骨细胞(ACh)使用定制的微孔平台(microwell -mesh)组装而成。微组织迅速积累均匀的软骨样细胞外基质(ECM),使其成为软骨缺损修复的潜在有用的构建块。软骨组织培养5或10 d后,在90%血清加10%二甲基亚砜(DMSO)或无血清商业冷冻保存培养基中冷冻保存。在解冻过程中,通过增加血清稀释以减少肿瘤性休克,使细胞活力最大化,然后在无血清培养基中洗涤和进一步培养。当用活/死活力染料评估时,解冻后的微组织表现出较高的活力,但相对于未冷冻的对照微组织,其即时代谢活性降低。为了进一步评估冻融微组织的功能,在14天的培养中评估其融合成连续组织的能力。培养5天的微组织融合性优于培养10天的微组织。关键是,冷冻保存的微组织在随后的14天培养中融合成连续组织的能力没有受到损害,这表明冷冻保存的微组织可以在软骨缺损部位融合。在2% O2气氛中进行汞化时,ECM的质量优于在20% O2气氛中进行汞化,这表明该工艺可能受益于接头内有限的氧气微环境。总之,软骨显微组织的低温保存是一种可行的选择,这种操作可以在不损害组织功能的情况下进行。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Tissue Engineering
Journal of Tissue Engineering Engineering-Biomedical Engineering
CiteScore
11.60
自引率
4.90%
发文量
52
审稿时长
12 weeks
期刊介绍: The Journal of Tissue Engineering (JTE) is a peer-reviewed, open-access journal dedicated to scientific research in the field of tissue engineering and its clinical applications. Our journal encompasses a wide range of interests, from the fundamental aspects of stem cells and progenitor cells, including their expansion to viable numbers, to an in-depth understanding of their differentiation processes. Join us in exploring the latest advancements in tissue engineering and its clinical translation.
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