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Purpose: To investigate the viability of cells collected with an in-line-suction autologous tissue collector from the tissue byproducts of arthroscopic anterior cruciate ligament (ACL) reconstruction, to characterize cells from different tissue types, and to identify mesenchymal stem cells., Methods: Patients aged 14 to 50 years with ACL injuries requiring arthroscopic reconstruction surgery were offered enrollment and screened for participation. In total, 12 patients were enrolled in the descriptive laboratory study. Arthroscopic byproduct tissue was collected with an in-line-suction autologous tissue collector from 4 intraoperative collection sites for each patient: ACL stump, ACL fat pad, notchplasty debris, and tunnel drilling debris. All tissue samples were digested using collagenase, and the derived cellular populations were analyzed in vitro, characterizing cellular viability, proliferative potential, qualitative multipotent differentiation capacity, and cell-surface marker presence., Results: An equivalent mass of arthroscopic byproduct tissue was taken from each of the 4 intraoperative collection sites (1.12-1.61 g, P  = .433), which all showed an average viability of at least 99.95% and high average total nucleated cells (≥1.37 × 10 7 cells/mL). No significant differences in collected mass ( P  = .433), cellular viability ( P  = .880), or total nucleated cells ( P  = .692) were observed between the 4 byproduct tissues. The byproduct tissues did exhibit significant differences in monocyte ( P  = .037) and red blood cell ( P  = .038) concentrations, specifically with greater values present in the ACL stump tissue. Cells from all byproduct tissues adhered to plastic cell culture flasks. Significant differences were found between colony-forming unit fibroblast counts of the 4 byproduct tissues when plated at 10 6 ( P  = .003) and 10 3 ( P  = .016) cells as the initial seeding density. There was a significant relationship found between both the starting concentration (χ 2  = 32.7, P < .001) and the byproduct tissue type (χ 2  = 30.4, P < .001) to the presence of ≥80% confluency status at 10 days. Cells obtained from all 4 byproduct tissues qualitatively showed positive tri-lineage (adipocyte, osteoblast, chondroblast) differentiation potential compared with negative controls under standardized in vitro differentiation conditions. Cells derived from all 4 byproduct tissues expressed cell-surface antigens CD105+, CD73+, CD90+, CD45-, CD14-, and CD19- (>75%), and did not express CD45 (<10%). There were no statistically significant differences in cell-surface antigens between the four byproduct tissues., Conclusions: This descriptive laboratory study demonstrated that cells derived from arthroscopic byproduct tissues of ACL reconstruction remain viable when collected with an in-line-suction autologous tissue collector and these cells meet the ISCT criteria to qualify as mesenchymal stem cells., Clinical Relevance: It is known that viable mesenchymal stem cells reside in byproduct tissue of anterior cruciate ligament reconstruction surgery (ACLR). Practical methods to harvest these cells at the point of care require further development. This study validates the use of an in-line-suction autologous tissue collector for the harvest of viable mesenchymal stem cells after ACLR., Competing Interests: The authors report the following potential conflicts of interest or sources of funding: This work was financially supported in part by a grant from Arthrex, Grant Number US19013. A.W.A. reports a grant from Arthrex, during the conduct of the study, and personal fees from Arthrex, outside the submitted work. E.A.B. reports a grant from Arthrex, during the conduct of the study, and personal fees from Arthrex, outside the submitted work. R.V.O. reports a grant from Arthrex, during the conduct of the study, and personal fees from Arthrex, outside the submitted work. S.E.J. reports a grant from Arthrex, during the conduct of the study, and personal fees from Arthrex, outside the submitted work. All other authors (J.J.C., C.A.R.) declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Full ICMJE author disclosure forms are available for this article online, as supplementary material., (© 2023 The Authors.)