Shajib Md Shafiullah, Futrega Kathryn, Franco Rose Ann G, McKenna Eamonn, Guillesser Bianca, Klein Travis J, Crawford Ross W, Doran Michael R
School of Biomedical Science, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia.
Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology, Brisbane, QLD, Australia.
J Tissue Eng. 2023 Jul 26;14:20417314231176901. doi: 10.1177/20417314231176901. eCollection 2023 Jan-Dec.
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% O atmosphere than a 20% O 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.
细胞和组织工程疗法的财务可行性可能取决于该疗法与大规模生产和冷冻保存的兼容性。在此，我们开发了一种用于3D软骨微组织大规模生产和冷冻保存的方法。软骨微组织由5000个人类骨髓间充质干细胞（BMSC）或5000个人类关节软骨细胞（ACh）分别使用定制的微孔平台（微孔网）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构建块。软骨微组织培养5天或10天，然后在90%血清加10%二甲基亚砜（DMSO）或商业无血清冷冻保存培养基中冷冻保存。通过逐步稀释血清以减少渗透性休克，然后洗涤并在无血清培养基中进一步培养，使细胞活力在解冻过程中最大化。当用活/死活力染料评估时，解冻后的微组织显示出高活力，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天的培养过程中评估它们融合成连续组织的能力。培养5天的微组织的融合优于培养10天的微组织。至关重要的是，冷冻保存的微组织在随后14天培养中融合成连续组织的能力并未受到损害，这表明冷冻保存的微组织可以在软骨缺损部位融合。当在2%氧气气氛中进行融合时，优质ECM优于在20%氧气气氛中，这表明该过程可能受益于关节内有限的氧气微环境。总之，软骨微组织的冷冻保存是一种可行的选择，并且这种操作可以在不损害组织功能的情况下进行。
