In vitro changes in human tenocyte cultures obtained from proximal biceps tendon: multiple passages result in changes in routine cell markers
- 505 Downloads
Results of in vitro cell models are commonly used to promote new therapies (e.g., platelet-rich plasma), and clinicians have to be aware of the specific limitations of such models. To gain a sufficient and effective cell load, many current in vitro models use cells multiplied through various passages. This is especially important in tendon-like cell (TLC) models, since native tendon tissue is not available unlimited and contains limited amount of tenocytes. The purpose was to determine the occurrence of phenotypic changes following extended monolayer culture of TLCs, according to cell-passage number.
Tendon samples were obtained from 15 healthy patients undergoing biceps tenodesis. Tendons were digested and cultured (monolayer) for six passages. Tendon-specific markers (collagens I and III, decorin, tenascin-C, and tenomodulin) and their histology were analyzed using gene expression and protein content assays. Native cells, the cells cultured and cells passaged one to six times were analyzed at each passage.
Gene expression of types I and III collagen of cultured TLCs significantly decreased after two passages. Gene expression of decorin, tenascin-C, and tenomodulin exhibited a trend of decreased gene expression with increased passage. Protein levels of types I and III collagen and decorin decreased after four passages.
The significant findings let conclude that tenocyte-like cells obtained from human LHB can be maintained in monolayer culture at low passages, before the signs of phenotypic drift are present. But researchers must be aware of rapid phenotypic drift at higher passage numbers. Therefore, only cells within the first 3 passages should be used as a precaution for in vitro monolayer cell models, and one has to be aware of the phenotypic changes if TLCs passaged multiple times are used. The clinical relevance of this data is that understanding of in vitro TLC models, and their limitations may finally help the clinician to judge the potential of experimental data of new biologic treatment options.
KeywordsTenocytes Cell passage Cell culture Phenotypic drift
The University of Connecticut Health Center/New England Musculoskeletal Institute has received direct funding, and material support for this study was provided by Arthrex Inc. (Naples, Fl). The company had no influence on study design, data collection or interpretation of the results. Augustus D Mazzocca receives research support and is a consultant for Arthrex, Inc.; all other authors, their immediate family, and any research foundation with which they are affiliated have not received any financial payments or other benefits from any commercial entity related to the subject of this article.
- 10.Maffulli N, Yao L, Bestwick C, Bestwick L, Aspden R (2008) Phenotypic drift in human tenocyte culture. J Bone Joint Surg Br 90-B (SUPP_III):494-aGoogle Scholar
- 18.Stoll C, John T, Endres M, Rosen C, Kaps C, Kohl B, Sittinger M, Ertel W, Schulze-Tanzil G (2010) Extracellular matrix expression of human tenocytes in three-dimensional air-liquid and PLGA cultures compared with tendon tissue: implications for tendon tissue engineering. J Orthop Res 28(9):1170–1177PubMedCrossRefGoogle Scholar
- 26.Zhang L, Tran N, Chen HQ, Kahn CJ, Marchal S, Groubatch F, Wang X (2008) Time-related changes in expression of collagen types I and III and of tenascin-C in rat bone mesenchymal stem cells under co-culture with ligament fibroblasts or uniaxial stretching. Cell Tissue Res 332(1):101–109PubMedCrossRefGoogle Scholar