The Upscale Manufacture of Chondrocytes for Allogeneic Cartilage Therapies.
The Upscale Manufacture of Chondrocytes for Allogeneic Cartilage Therapies.
复制标题
DOI：
10.1089/ten.tec.2023.0037
复制
发表时间：
2023-09
期刊：
影响因子：
--
通讯作者：
Wright KT
中科院分区：
文献类型：
--
作者：
Hulme CH;Garcia JK;Mennan C;Perry J;Roberts S;Norris K;Baird D;Rix L;Banerjee R;Meyer C;Wright KT
需要开发同种异体软骨细胞疗法，以允许更多的人接受软骨修复的细胞疗法，并减少目前两阶段自体手术的负担和成本。使用生物反应器大规模制造软骨细胞可以帮助提供现成的同种异体软骨细胞疗法，在一次生产过程中可以生产许多剂量。在这项研究中，我们评估了用于成人软骨细胞制造的符合生产实践的中空纤维生物反应器(Quantum®)。从膝关节置换术后的软骨(n = 5)中分离软骨细胞，在含有10%胎牛血清或5%人血小板裂解液的组织培养塑料上进行单代扩增。然后将添加了HPL的培养物在量子生物反应器中进行扩增，以便进一步传代。HPL或FBS中的匹配、平行培养在Tcp上维持。对所有培养条件下的软骨细胞进行生长动力学、细胞形态、免疫学特性、成软骨潜能(软骨细胞颗粒分析)和单个端粒长度分析。软骨细胞量子扩增后，接种10.2 ± 3.6 × 10 6细胞，在8.4 ± 1.5d内获得86.4 ± 38.5 × 10 6细胞。这与量子生物反应器中3.0 ± 1.0的种群倍增有关，而在添加HPL和FBS的培养液中，TcP上的种群倍增分别为2.1 ± 0.6和1.3 ± 1.0。Quantum和Tcp扩增的细胞保持了同等的软骨潜能和间充质细胞标志物的免疫图谱，只有整合素标志物CD49a在Quantum扩增后降低。量子扩增的软骨细胞表现出与匹配的HPL-TCP群体相同的软骨形成潜力(通过形成和维持成软骨颗粒的能力进行评估)。然而，与FBS培养相比，HPL的制造降低了软骨形成潜力，增加了整合素CD49b、CD49c和CD51/61的细胞表面阳性表达。与匹配的TCP培养相比，软骨细胞的量子扩张没有导致17P端粒长度缩短。这项研究表明，在量子中空纤维生物反应器中可以制造大量的成年软骨细胞。这种快速、高档的扩增与相匹配的TCP扩增相比不会改变软骨细胞的表型。因此，Quantum为临床提供了一种有吸引力的软骨细胞制造方法。然而，添加HPL的培养液用于软骨细胞的扩增在保留软骨形成能力方面可能是不利的。据我们所知，这是第一项在符合制造规范的中空纤维生物反应器(Quantum®)中制造成人软骨细胞的研究。我们提供的证据表明，可以使用这种方法制造软骨细胞，同时保留与在匹配的组织培养塑料条件下扩张的软骨细胞类似的特性。高端的Quantum®Expansion可能为开发同种异体软骨细胞疗法提供了一种合适的方法。
Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of the current two-stage autologous procedures. Upscale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. In this study, we assess a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum®) for adult chondrocyte manufacture. Chondrocytes were isolated from knee arthroplasty-derived cartilage (n = 5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL-supplemented cultures were then expanded in the Quantum bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterized in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays), and single telomere length analysis. Quantum expansion of chondrocytes resulted in 86.4 ± 38.5 × 106 cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 106 cells. This related to 3.0 ± 1.0 population doublings in the Quantum bioreactor, compared with 2.1 ± 0.6 and 1.3 ± 1.0 on TCP in hPL- and FBS-supplemented media, respectively. Quantum- and TCP-expanded cultures retained equivalent chondropotency and mesenchymal stromal cell marker immunoprofiles, with only the integrin marker, CD49a, decreasing following Quantum expansion. Quantum-expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture, however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c, and CD51/61 compared with FBS cultures. Quantum expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum hollow-fiber bioreactor. This rapid, upscale expansion does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavorable in terms of retaining chondrogenic capacity. This is the first study, to our knowledge, to manufacture adult chondrocytes in a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum®). We provide evidence that chondrocytes can be manufactured using this methodology while retaining comparable properties to chondrocytes expanded in matched tissue culture plastic conditions. Upscale Quantum® expansion may provide an appropriate method for developing allogeneic chondrocyte therapies.
影响因子：
6
作者：
Coccè V;La Monica S;Bonelli M;Alessandri G;Alfieri R;Lagrasta CA;Madeddu D;Frati C;Flammini L;Lisini D;Marcianti A;Parati E;Paino F;Giannì A;Farronato G;Falco A;Spaggiari L;Petrella F;Pessina A
通讯作者：
Pessina A