Cell culture techniques have been developed to replicate and maintain the chondrocyte phenotype in vitro for clinical and cartilage tissue engineering applications [1]. When chondrocytes are isolated from cartilage tissue and grown in monolayer culture, they undergo a reversible dedifferentiation process whereby the cells lose their chondrocyte phenotype with a change in morphology from a round shape to fibroblastic-like [18, 19]. This physical change in the cells is accompanied by a downregulation of collagen II expression and several chondrogenic genes including SZP, BMP-2, TGFb1, FGFR3, COMP, aggregan, collagen IX, and SOX 9. Furthermore, there is an upregulation of collagen I, collagen X, collagen III, and tenascin and versican gene expression [20–22]. However, dedifferentiation can be reversed by transferring the cells from a monolayer culture to a three-dimensional culture such as an agarose or alginate suspension culture [18, 23–25]. Upon redifferentiation there is an upregulation of chondrogenic genes [26–28]. The cell and molecular mechanisms which control the dedifferentiation and re-differentiation of chondrocytes in cell culture have not been fully elucidated; however it has been found that the actin stress fibers which are involved in cell spreading play a role [29, 30]. The addition of growth factors of PDGF-BB, TGFβ1, and FGF2 has been found to increase cell proliferation and aid the recovery of chondrocyte phenotype in vitro [31, 32]. These in vitro properties of chondrocyte cultures and growth factors that affect them have been harnessed to develop cell culture protocols that optimize chondrocyte phenotype toward cartilage repair.

The purpose of this protocol is not to provide enough detail to replicate but rather have the reader develop an appreciation for the steps, processes, and reagents involved in chondrocyte cell culture. More detailed protocols are available elsewhere [1, 33–35]. In the USA, culture and manufacture of Carticel^® is based on the protocol originally developed by Tubo and Binette [1, 11]. A cartilage biopsy of about 200–300 mg is retrieved from a non-weight-bearing area preferably the intercondylar notch in an outpatient arthroscopic procedure by the treating surgeon. The biopsy is then transported in serum-free DMEM to the cGMP laboratory for processing and cell culture expansion [11]. Within 48 h of arrival in the laboratory, the biopsy is treated with enzymatic digestion and minced in to 0.5 mm to isolate the chondrocytes [1]. The digestion medium consists of 0.1% w/v collagenase solution in DMEM at 37 °C. The minced cartilage in digestion medium is transferred into a centrifuge tube and then put into an incubator at 37 °C overnight. The following day the digested cartilage is removed from the incubator, supernatant collected, and transferred to another tube. The enzyme is inactivated by adding more media and the cells counted with a hemocytometer. The cell suspension is then centrifuged to concentrate the cells and the cell pellet added with media (DMEM with 10% FBS) into a flask for expansion and placed in incubator at 37 °C [1]. The cell media is replaced every 2–3 days, and the cells are passaged once they reach 80–90% confluence. The passaged cells are diluted 1:10 in cell culture media (DMEM with 10%FBS) and seeded for further monolayer expansion [1]. Cell culture expansion for 2–3 weeks is needed to produce approximately 12 million cells. Typically, cells are cryopreserved after initial cell expansion and retrieved for further expansion after an order made. At which point, 2–3 days before the patient’s surgery for implantation, the cells are processed into shipping vials. Several assays are performed to verify the final product including sterility testing, Mycoplasma, viability, identity, potency, and visual inspection. The BacT/Alert system is used for sterility testing [36]. Cell identity assays involving gene expression analysis distinguish chondrocytes from synoviocytes and dermal fibroblasts [37]. There are different protocols available for chondrocyte culture and expansion, and many of those used for commercial production of chondrocytes for clinical applications are propriety. In general, differences in protocols will include the type of cell culture medium used, use of serum, growth factors, and other supplements [20].

To date there have been two culture expanded cell therapy products that have been approved in the U.S.A by the FDA for cartilage repair, Carticel® and MACI®. There are several products that use culture-expanded chondrocytes that have been approved in other countries, are currently in clinical development, or have terminated their clinical trial or manufacture. ChondroCelect^® was the first cell-based product to be approved in Europe. The company has ceased manufacture of the product. It is a two-stage procedure where cultured autologous chondrocytes are implanted in a suspension under a periosteal flap or membrane. MACI^® (matrix-induced autologous chondrocyte implant) is a third-generation ACI product that was marketed in the EU. The manufacturer of the product, Vericel Corporation, received approval for MACI® in the USA in December 2016. MACI^® consists of autologous cultured chondrocytes seeded onto a 14.5 cm² resorbable type I/III collagen membrane at a density of 500,000 to 1 million cells per cm² which can be trimmed by the surgeon according to the size and shape of defect [38]. NOVOCART 3D^® is also third-generation ACI product that is currently undergoing clinical trials in the USA for articular cartilage repair of the knee. The product consists of autologous chondrocytes seeded on a bovine collagen type 1/chondroitin sulfate sponge. Prior to seeding the chondrocytes are cultured in autologous serum for one passage without the use of antibiotics. The duration from isolation of the chondrocytes to completed product is 3 weeks. NeoCart^® consists of autologous cultured chondrocytes on a bovine type I collagen scaffold. The cell culture involves a biomimetic bioreactor with hydrostatic pressure which is applied during the first 6 days of culture; hypoxia (2% O₂) and perfusion at 5 μL/min are also part of the bioreactor culture process. It is believed that these conditions increase S-GAG production and cell proliferation. It takes approximately 67 days from biopsy before the implant is available.

There is a great variation in the initial number of MSCs depending on the source [39]. Cell culture can be used to expand the low populations of MSCs found in some tissues in order to generate sufficient quantities for therapeutic use. For example, MSCs within the bone marrow comprise just a small portion, approximately 0.001–0.1%, of the mononuclear cell population within the bone marrow [50–52]. Furthermore, MSCs are found within a milieu of heterogeneous tissue, and cell culture enables the enrichment of MSC populations. MSCs are capable of self-renewal and can be expanded considerably in vitro; however at about five or more passages in culture, MSCs start to exhibit changes in morphology, reduced proliferation, and shortening of telomeres and change the profile of MSC markers [53–57]. The addition of FGF2 has found to extend the ability of MSC cultures to maintain their self-renewing and differential potential for longer [58].

There have been several approaches reported to direct the differentiation of MSCs into chondrocytes in vitro. The most common protocols for chondrogenic differentiation involve pellet cultures [59, 60] with various media components and growth factors. In pellet culture MSCs undergo cell condensation as well as cell to cell and cell to extracellular matrix interactions [61, 62]. This is followed by a highly proliferative stage whereby the cells start to produce collagen type 2 and other components of the ECM [63–68]. In the terminal stages of differentiation, the cells become rounded and begin to exhibit signs of hypertrophy including the expression of collagen type X and MMP13. The different stages of chondrocyte differentiation are controlled by transcription factors including SOX5, SOX6, and SOX 9 [69–72]. The addition of key growth factors to the cell medium is essential for the differentiation process. TGFβ is a well-established growth factor known to induce chondrogenesis [60, 73]. Some protocols also include BMPs [74–76] or IGF1 [59] as growth factors. In addition, environmental cues such as mechanostimulation [77] and hypoxia [78, 79] have been shown to be modulators of MSC chondrogenesis. There are several methods to assess chondrogenesis in MSC cultures. The production of ECM is associated with a substantial increase in cell mass that can be assessed by changes in the weight of the cultures over time. The production of COL2A1 can be measured by gene expression with increase in expression from 100- to 1000-fold in chondrogenic cells compared to the primary MSC cultures. The generation of proteoglycans can be examined by histological staining with alcian blue, toluidine blue, or safranin O [80]. The tendency of in vitro MSC-derived chondrocytes to undergo hypertrophy and subsequent osteochondral ossification is undesirable. This transformation into hypertrophic chondrocytes triggered by the endochondral pathway is marked by an upregulation of osteogenic genes [81] and collagen type X [82]. There is still room for improvement in terms of understanding the basic biology of MSCs and optimizing culture conditions to ensure that tissue-engineered products provide high-quality and durable tissue.

Currently there is no FDA-approved product that uses MSC-derived cells for cartilage repair or any other orthopedic indication. However, there have been several clinical reports and clinical trials using MSCs. None of the reported clinical applications of cultured MSCs for cartilage repair use directed differentiation into chondrocytes in vitro. Rather, the cultured multipotent MSCs are directly applied onto a scaffold, gels, and other carriers and surgically implanted into the defect, where presumable in vivo signals within the defect direct the differentiation of the MSCs for repair.