Purpose: Blood-derived products are known to be involved in musculoskeletal regeneration by promoting cell proliferation and modulating inflammation. By altering the cell-hostile microenvironment we are pursuing to stop or even reverse the degenerative process in the joints of osteoarthritic patients. Hyperacute serum is a blood-derived product that retains the regenerative potential of platelet rich plasma (PRP) overcoming its variability disadvantages. Hyperacute serum obtained by squeezing a fibrin clot and its composition has been compared to PRP, showing that it better resembles cellular and molecular concentrations of in vitro and in vivo samples. To expand the potential therapeutic use, a new formulation method as lyophilized powder was established. This freeze-dried format will enable allogenic therapy with a highly regenerative, standardized and stable blood product. In contrast to freshly isolated hyperacute serum, which allows for autologous therapy, where the donors and the patients are the same individuals, most of the time elderly OA patients; the allogenic serum is a nutrient-enriched version as it is obtained from young healthy donors. Moreover, it is manufactured by pooling multiple donor´s serum, which solves the problem of donor variability. Hyperacute serum in lyophilized format can be stored for longer periods of time, therefore, administration to patients is facilitated. Two different manufacturing processes lead to the development of two different versions of lyophilized hyperacute serum. Our aim is to test the regenerative capacities of the two new lyophilized hyperacute sera in the context of osteoarthritis (OA) alone and in combination with hyaluronic acid (HA). Methods: Two-dimensional (2D) cultures of primary chondrocytes were kept in culture under different supplementation conditions, including three versions of hyperacute serum (one freshly isolated and two lyophilized), PRP, FCS and combinations of hyperacute serum+HA. These 2D cultures were analyzed with the XTT viability assay. 3D chondrogenic spheroids from primary-isolated chondrocytes were developed and kept in culture for 1 week within the listed culture conditions. In addition to XTT viability analysis , collagen type II and aggrecan production and secretion within the extracellular matrix, confocal microscopy together with immunofluorescence were applied. Results: XTT assays (sample size=5) of the 2D cultures were performed at the following time points: day 3, day 5 and day 7. When comparing the different formats of hyperacute serum at day 3, we have observed that 10% lyophilized extra-filtered hyperacute serum promotes cell viability in a comparable level to 10% FCS (1,87 and 1,57 a.u. respectively, ns). Moreover, increasing the concentration of this hyperacute serum to 20% leads to 2,27 a.u.; however, this increase did not reach the level of statistical significance. In previous publications, it was shown that freshly isolated hyperacute serum had a high performance in promoting cell viability as well as in affecting gene expression of genes that play a role in osteoarthritis, by holding back inflammation (TNFα, Il-1β, Il-1ra), promoting tissue regeneration (COL1A, osteonectin) and preventing bone degradation (RANKL). However, lyophilized extra-filtered hyperacute serum did not present the same strong effect. We hypothesize that due to the further filtration process, nutrients including growth factors were removed, which had a clear impact in promoting cell viability. Nevertheless, filtered lyophilized hyperacute serum still showed a comparable effect as FCS, known to be a gold standand in cellular supplementation. Due to the loss of nutrient supply, a non-filtered lyophilized hyperacute serum with a different manufacturing process was produced. Same experiments were repeated demonstrating that non-filtered lyophilized hyperacute serum shows a significant promotion of cell viability (2,93 a.u., P=0,0313), similarly to freshly isolated hyperacute serum or PRP (3,26 a.u. and 3,15 a.u., respectively; ns.). When testing the effect of HA alone (2,19 a.u., ns) or in combination with extra-filtered or non-filtered lyophilized hyperacute sera, it showed no significant differences in cell viability due to a possible added effect on such hypercute sera (1,83 a.u. and 3,39 a.u., respectively; ns.). Further gene expression analysis need to be performed in order to better understand the mechanisms involved in each of the conditions. XTT data from days 5 and 7 of culture supported the findings concerning HA and the significant difference between the two lyophilized versions of hyperacute sera; however, it revealed higher cell viability when cultures were supplemented with 10% PRP and 10% freshly isolated hyperacute serum. Confocal microscopy of 3D chondrogenic pellets has shown that production and secretion of collagen type II and aggrecan, two main structural proteins of the cartilage, increased in 3D chondrogenic spheroids supplemented with the different formulations of hyperacute sera as well as FCS. Conclusions: Non-filtered lyophilized hyperacute serum maintains the supplementation capacity in 2D and 3D chondrogenic cultures, as shown in the past by the freshly isolated format. The implementation of hyaluronic acid to such blood-derived products does not show, for now, any added effect. Non-filtered lyophilized hyperacute serum is a stable and standardized blood product with regenerative potential in the context of OA.