Mesenchymal Stromal Cells

Mesenchymal stromal cells were derived from lipoaspirates obtained from consenting donors and clinical trial patients with approval from the Mayo Clinic Institutional Review Board (IRB) as previously described [12 (link), 29 (link)]. After harvesting, fat tissue was digested with collagenase (type I at 0.075 %; Worthington Biochemicals) for 1.5 h at 37 °C. The stromal vascular fraction was isolated by low speed centrifugation (400 g for 5 min), the supernatant was removed, and the cell pellet was rinsed with PBS and passed through a cell strainer (70 μm) (BD Biosciences). Buffered ammonium solution (154 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) was used to lyse erythrocytes. The resulting cell fraction was plated onto tissue culture flasks in standard culture medium (advanced MEM) with 5 % hPL (Mill Creek Life Sciences), 2 mM L-glutamine (Invitrogen), and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin) and maintained 37 °C in 5 % CO₂ at a cell density of 1.0–2.5 × 10³ cells/cm².
Prior to cryopreservation, freshly isolated AMSCs were harvested for flow cytometric analysis (pre-thaw samples), and the remaining cells were frozen in 1 ml aliquots of up to 20 × 10⁶ cells/mL with CryoStor CS10 Cryopreservation Medium (BioLife Solutions, Stem Cell Technologies) and stored in liquid nitrogen. Post-thaw samples were recovered from cryopreservation by placing the vial in a 37 °C water bath to thaw, then transferred to 10 ml of growth media and centrifuged for 5 min at 500 g. Cells were resuspended in growth media and approximately 1 × 10⁶ cells were harvested for flow cytometric analysis. The remaining thawed cells were plated in T-175 cm² flasks at a density of up to 3000 cells/cm² and cultured for 4 days at 37 °C in 5 % CO₂. After 4 days’ culture, cells were harvested for flow cytometric analysis.

Centers were requested to report patients receiving cellular therapies other than HCT. Hematopoietic advanced cellular therapies were defined as infusion of cells undergoing substantial manipulation after collection, either selection and/or expansion, or genetic modification and thus qualify as investigational or approved advanced therapy medicinal products (ATMPs) according to Regulation (EC) N° 1394/2007. In this context, “substantial” should be understood as referring to the definition included in the Regulation and subsequent regulatory documents and may not reflect the workload assumed by cell processing facilities working in conjunction with clinical programs. Depending on their nature and indications, hematopoietic cellular therapies may be designed to replace or to complement HCT. Administration of non-substantially manipulated hematopoietic cells, such as transplantation of CD34+ selected hematopoietic stem cells were counted as HCT and not as cellular therapy [18 (link)]. Similarly, un-manipulated lymphocyte infusions post-HCT were counted as donor lymphocyte infusions (DLI) and not as cellular therapy. Hematopoietic cellular therapies include immune effector cells as defined in FACT-JACIE standards for Hematopoietic Cellular Therapy: “A cell that has differentiated into a form capable of modulating or effecting a specific immune response.” This definition covers CAR-T cells and forms the basis for accreditation requirements in recent EBMT-JACIE recommendations [17 , 19 (link)].
Hematopoietic cellular therapies were categorized as chimeric antigen receptor T cells (CAR -T); in vitro selected/and or expanded T cells or cytokine activated, such as virus specific T cells; cytokine-induced killer cells (CIK); regulatory T cells (TREGS); genetically modified T cells other than CAR-T; natural killer cells (NK); dendritic cells; mesenchymal stromal cells; in vitro expanded CD34+ cells; and genetically modified CD34+ cells. This survey did not include cells from sources other than hematopoietic tissue. On the other hand, gene therapy protocols, such as those used to treat thalassemia or SCID were included, however numbers have remained low.