The function of connective tissues depends on the physical and biochemical properties of their extracellular matrix (ECM), which are in turn dictated by ECM protein composition. With the primary objective of obtaining quantitative estimates for absolute and relative amounts of ECM proteins, we performed a systematic review of papers reporting protein composition of human connective tissues. Articles were included in meta-analysis if they contained absolute or relative quantification of proteins found in the ECM of human bone, adipose tissue, tendon, ligament, cartilage and skeletal muscle. We generated absolute quantitative estimates for collagen in articular cartilage, intervertebral disk (IVD), skeletal muscle, tendon, and adipose tissue. In addition, sulfated glycosaminoglycans were quantified in articular cartilage, tendon and skeletal muscle; total proteoglycans in IVD and articular cartilage, fibronectin in tendon, ligament and articular cartilage, and elastin in tendon and IVD cartilage. We identified significant increases in collagen content in the annulus fibrosus of degenerating IVD and osteoarthritic articular cartilage, and in elastin content in degenerating disc. In contrast, collagen content was decreased in the scoliotic IVD. Finally, we built quantitative whole-tissue component breakdowns. Quantitative estimates improve our understanding of composition of human connective tissues, providing insights into their function in physiology and pathology.

MAMMALIAN CELL CULTURE is an essential tool to examine cell function in a controlled environment. Cell culture relies on the assumption that the behavior of cells in vitro is fundamentally similar to their behavior as part of a tissue within an organ of a multicellular organism. Nevertheless, the difficulties with translation of in vitro results into animal models and beyond persist (4, 25). Significant problems with current cell culture methodology have been identified, including the lack of proper mechanical environment (21), hyperoxic conditions of culturing at atmospheric PO 2 (14), and the accumulation of waste products (13). In addition, alternatives to media supplementation with undefined animal serum are being actively developed (2). We now bring attention to the fact that the very basic components of cell culture medium-electrolyte and carbohydrate levels-do not properly model the physiological environment.Modern culture media were developed over 60 years ago, starting with medium 199 (M199) in 1950 and Harry Eagle's minimum essential medium (MEM) in 1959 (11,19). While media formulations tailored for specific applications are continuously developed, the majority of in vitro cell culture experiments employ standard media formulations (2). According to a Google Scholar search, 73,200 papers contained the search term "culture medium" in 2016, including 31,300 papers that used Dulbecco's modified Eagle's medium (DMEM), 24,700 with RPMI 1640, 6,480 with MEM and 480 with its Alpha modification; and 1,650 with M199. Thus, these four media formulations were used in~90% of published in vitro studies.We compared concentrations of Na ϩ , K ϩ , Ca 2ϩ , Mg 2ϩ , Cl Ϫ , PO 4 2Ϫ , HCO 3 Ϫ , SO 4 2Ϫ , and glucose in human plasma (16, 29a), mouse plasma (12,15,24,29), and human cerebrospinal fluid (CSF) (28) to concentrations of these components in DMEM, RPMI 1640, MEM, and M199 [Sigma-Aldrich product information ( 27)]. We attempted to obtain information on electrolyte and glucose levels in serum-free media from Sigma-Aldrich, ThermoFisher, and Lonza; however, company representatives were unable to provide us with this detailed information, citing proprietary reasons. The representative of Sigma-Aldrich indicated that their serum-free media are based, at least in part, on DMEM, suggesting that deviations in electrolyte and carbohydrate concentration may persist in those media.Notably, none of the examined media fully adhere to physiological values of electrolytes and carbohydrates (Table 1).