Hypothermic Preservation of Adipose-Derived Mesenchymal Stromal Cells as a Viable Solution for the Storage and Distribution of Cell Therapy Products

Abstract

Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily burdened process. Encapsulation at hypothermic temperatures (e.g., 2-8 °C) could be a feasible alternative. Adipose tissue-derived mesenchymal stromal cells (MSC(AT)) expanded using fetal bovine serum (FBS)- (MSC-FBS) or human platelet lysate (HPL)-supplemented mediums (MSC-HPL) were encapsulated in alginate beads for 30 min, 5 days, and 12 days. After bead release, cell recovery and viability were determined to assess encapsulation performance. MSC identity was verified by flow cytometry, and a set of assays was performed to evaluate functionality. MSC(AT) were able to survive encapsulated for a standard transportation period of 5 days, with recovery values of 56 ± 5% for MSC-FBS and 77 ± 6% for MSC-HPL (which is a negligible drop compared to earlier timepoints). Importantly, MSC function did not suffer from encapsulation, with recovered cells showing robust differentiation potential, expression of immunomodulatory molecules, and hematopoietic support capacity. MSC(AT) encapsulation was proven possible for a remarkable 12 day period. There is currently no solution to completely replace cryopreservation in CGT logistics and supply chain, although encapsulation has shown potential to act as a serious competitor.

Keywords: cell encapsulation; fetal bovine serum; hematopoietic support assay; human platelet lysate; hypothermic temperatures; mesenchymal stromal cells; xenogeneic-free.

Figure 1

Study design. Three different adipose tissue-derived mesenchymal stromal cell (MSC(AT)) donors were expanded in fetal bovine serum (FBS) or human platelet lysate (HPL)-supplemented expansion medium in standard tissue culture plastic. MSC(AT) were encapsulated in alginate beads and kept at temperatures between 10 and 20 °C after reaching desired temperatures. MSC(AT) were left encapsulated during three different time periods: 30 min (D0), 5 days (D5), and 12 days (D12). Cells were then released and subjected to different characterization assays and compared with non-encapsulated MSC(AT). Cell retainment and survival during encapsulation, MSC identity and functional immunophenotype, MSC tri-lineage differentiation potential, metabolic activity, and hematopoietic support capacity were determined and compared between timepoints.

Figure 2

Cell encapsulation performance and MSC(AT) metabolic analysis. (A) Cell recovery from alginate beads after 30 min (D0), 5 days (D5), and 12 days (D12) for MSC-FBS (blue) and MSC-HPL (red). (B) Cell viability of MSC(AT) before encapsulation (Non) and after their release from encapsulation at D0, D5, and D12. (C) Glucose and lactate concentration profiles. (D) Glucose (left) and lactate (right) profile regression modelling, including the fitting of first-order regressions with the presentation of the equation and coefficient of determination (R²). (E) Molar glucose consumption and lactate production rates. (F) Specific molar glucose consumption and lactate production rates at the various encapsulation timepoints. (Three MSC(AT) donors; mean ± SEM; * p < 0.05, ** p < 0.01.)

Figure 3

Differentiation potential and immunophenotype of MSC(AT) before and after encapsulation. (A) Map of MSC(AT) tri-lineage differentiations showing successful differentiation in every timepoint. The representative images include osteogenic (left), adipogenic (center), and chondrogenic (right) stainings. (B) Positive and negative identity marker expressions for MSC-FBS (left) and MSC-HPL (right) (%). (C) Representative MSC(AT) marker expressions for a defined encapsulation timepoint. Dotplots containing stained cells (orange) were overlaid with the unstained control (dark grey) for homogeneous populations with no subpopulations identified (first, third, and fourth row). Marker expressions that led to MSC(AT) positive subpopulations were gated in contour plots (second row). Scale bar: 100 μm; Non—non-encapsulated; SSC—Side scatter; MFI—median fluorescence intensity (three MSC(AT) donors; mean ± SEM).

Figure 4

Characterization of MSC(AT) immunosuppression potential and clonogenic ability. (A) Subpopulation immunosuppressive and clonogenic marker expressions for MSC-FBS (left) and MSC-HPL (right). (B) Homogeneous immunosuppressive and clonogenic populations with marker percentage and median fluorescence intensity (MFI) levels for MSC-FBS (left) and MSC-HPL (right). The interconnected dots are the marker percentage and the bars are the MFI. (C) MFI analysis for motility (CD10), translocation (CD54), and hematopoietic support-related (CD146) markers for MSC-FBS (left) and MSC-HPL (right). Non—non-encapsulated; three MSC(AT) donors; mean ± SEM.

Figure 5

Hematopoietic support assay for MSC(AT) potency/function. (A) Experimental layout. Non-encapsulated and released MSC(AT) are replated as a feeder layer to investigate their hematopoietic support capacity. Umbilical cord blood-derived hematopoietic stem and progenitor cells (HSPC(CB)) were isolated via magnetic activated cell sorting (MACS) and seeded onto the MSC(AT) feeder layer. After 7 days in a co-culture setting, expanded HSPC(CB) were harvested and analyzed concerning their cell number, immunophenotype, metabolic activity, and differentiation potential using colony forming unit (CFU) assay. (B) Mean fold change (FC) in total nucleated cell (TNC) number after HSPC(CB) expansion normalized to the control condition (HSPC(CB) expanded without an MSC(AT) feeder layer (No FL). (C) Glucose (top) and lactate (bottom) concentration profiles for co-cultures of HSPC(CB) and MSC-FBS (left) and MSC-HPL (right). (D) Glucose consumption (left) and lactate production (right) rates during hematopoietic expansion. No FL—the control condition without an MSC(AT) feeder layer; Non—non-encapsulated; three MSC(AT) donors; mean ± SEM.

Figure 6

Immunophenotype and clonogenic potential (CFU) of hematopoietic stem/progenitor cells co-cultured with MSC(AT). (A) Representative dotplots showing the gating strategy used for the identification of different HSPC populations before expansion (Pre-Exp) and after expansion using released (D0, D5, and D12) or non-encapsulated (Non) MSC(AT) as feeder layers. Live HSPC were gated on forward scatter (FSC) versus side scatter (SSC) followed by the use of a viability dye. Then, CD34 expression was identified (top) and CD45RA and CD90 expression were also investigated to explore the remaining populations (bottom). (B) FC of normalized CD34⁺ (relative to the control No FL) (left), CD34⁺CD45RA⁻ (center), and CD34⁺CD45RA⁻CD90⁺ (right). (C) Percentage of CD34 expression (left), CD34⁺CD45RA⁻ (center), and CD34⁺CD45RA⁻CD90⁺ (right). (D) Quantification of CD34 loss after expansion. Mean fluorescence intensity (MFI) of CD34⁺ expression was quantified and normalized using the width of the positive CD34 population. (E) CFU population percentage. Neglectable burst-forming unit-erythroid (BFU-E) led mainly to two populations, including colony forming-unit granulocyte (CFU-GM) and colony forming-unit multilineage (CFU-Mix). (F) FC in total CFU number after HSPC expansion using FL from encapsulated and non-encapsulated MSC(AT) which were previously expanded in FBS or HPL supplemented medium. No FL—control condition without an MSC(AT) feeder layer; Non—non-encapsulated; SSC—Side scatter; LL—Lower limit; UL—Upper limit; three MSC(AT) donors; mean ± SEM, * p < 0.05, ** p < 0.01).

Figure 7

Heatmap score of MSC(AT) encapsulation. Key variables were put side-by-side to perform a comprehensive comparison between encapsulation timepoints for MSC-FBS (top) and MSC-HPL (bottom). Each variable was individually normalized using the value of non-encapsulated cells or D0 when non-encapsulated cells were not available. Differentiation was set to 1 for every timepoint as every differentiation was successful. Non—non-encapsulated; Glu—Glucose; Lact—Lactate; Norm—Normalized; FC—Fold Change; MFI—Median Fluorescence Intensity; n.a.—not applicable; three MSC(AT) donors; mean.