Study design
We formulated several xeno-free hPL-based cryopreservation solutions and evaluated their efficacy for cryopreserving ATMPs, including bioengineered tissue. For the latter, we tested cellularized NFAHs, currently used at the Unidad de Producción y Reprogramación Celular (UPRC, Seville, Spain) to treat burns. We also tested the following cell types commonly used in cell therapy: bone marrow-derived mesenchymal stem cells (BM-MSCs), human dermal fibroblasts (FBs) and neural stem cells (NSCs). ihPL and low-DMSO formulations were included in some of the solutions with the objective of providing additional safety and security. The study was performed in two Good Manufacturing Practice (GMP)-grade laboratories that manufacture and cryopreserve ATMPs for clinical use. Each GMP laboratory followed their standard methods based on previous experience (e.g., standard cryopreservation solution and number of cells) for each tissue/cell type for comparative analysis.
Generation Of Cellularized Nanostructured Fibrin Agarose Hydrogels
NFAHs are artificial tissues that are used for diverse clinical applications depending on the encapsulated cell type. For the present study, the NFAH contained FBs and was generated as previously described [11]. Briefly, 4.16 ml of human plasma obtained from healthy blood donors was added to 7.5 × 105 FBs resuspended in Dulbecco's Modified Eagle´s Medium (DMEM; Sigma Aldrich, St. Louis, MO) supplemented with gentamicin (20 µg/ml) (Normon, Madrid, Spain) and 83 µl of tranexamic acid (Amchafibrin® 500 mg; Rottapharm, Milan, Italy), as an anti-fibrinolytic agent. We then added 0.25 ml of 2.2% type VII-agarose (Sigma-Aldrich) in PBS (Sigma-Aldrich). When the temperature of the solution fell to < 40ºC, 300 µl of 10% calcium chloride (B.Braun, Melsungen, Germany) was added to support the fibrin polymerization reaction. Aliquots (5 ml) were placed in 24-mm diameter Transwells (Corning, Corning, CA) and allowed to solidify at 37°C for 2 hours. Subsequently, the wells were filled with DMEM (Sigma-Aldrich) supplemented with 5% hPL and 2 U/ml heparin (Rovi, Madrid, Spain) to prevent unwanted clotting. Fibrin agarose hydrogels were kept at 37°C for 12 days.
We then applied the nanostructuring technique, as described [11] (Fig. 1A). Briefly, we prepared an extra-thick western blotting filter paper (ThermoFisher Scientific, Waltham, MA) with a 10‐µm nylon net filter on top (Merck Millipore, Burlington, MA) to prevent adherence, and the hydrogel was placed over the filter. Then, another nylon net filter/blotting paper was sandwiched over the hydrogel. A flat glass weighting 0.25 kg was then quickly positioned on top for 1 min and 40 s for compression. Artificial tissues were manufactured at the UPRC (Seville, Spain).
Cell Lines
BM-MSCs were cultured and cryopreserved at the Unidad de Terapia Celular, Hospital Universitario Reina Sofía, (Córdoba, Spain) in alpha-minimum essential medium (αMEM; Lonza, Milan, Italy) supplemented with 13% (v/v) FBS (Gibco/Invitrogen, Carlsbad, CA), 2 mM Ultraglutamine 1 (Lonza) and 1 ng/ml fibroblast growth factor (Miltenyi Biotec, Bergisch Gladbach, Germany).
FBs were cultured at the UPRC (Seville, Spain) in DMEM supplemented with 10% (v/v) FBS (Sigma-Aldrich), 0.1 mM non-essential amino acids (NEAA; Sigma-Aldrich), 2 mM Glutamax (Gibco/Invitrogen) and 100 µg/ml gentamicin (Sigma-Aldrich).
NSCs [12] were cultured at the UPRC (Seville, Spain) in DMEM-F12 (ThermoFisher Scientific), 0.1 mM NEAA, 100 IU penicillin/100 µg/ml streptomycin (Sigma-Aldrich), 2 µg/ml heparin (Rovi), 1% N2, 1×B27 (both from ThermoFisher Scientific), 20 ng/ml FGF (Miltenyi Biotec), 20 ng/ml epidermal growth factor (Peprotech, Pcky Hill, NJ), and 10 ng/ml leukemia inhibitory factor (Miltenyi Biotec).
Testing Hpl-based Solutions
Based on a bibliographic study and our laboratory experience, we developed different formulations to evaluate cryopreserving of NFAHs and commonly used cells. hPL and ihPL were produced as described [10].
Five different hPL-based cryopreservation solutions (Ti1, Ti2, Ti3, Ti4 and Ti5) were developed and tested for cryopreserving NFAHs and were compared against two control solutions (the standard cryopreservation solution at our laboratory and a commercially available one). The conditions and reagents used for the bioengineered tissue cryopreservation process are described in Table 1 and Additional file 1. All cryoprotective solutions were kept on ice until use. NFAHs were placed into a Maco Biotech Freezing EVA bag (Macopharma Biotech, Tourcoing, France) containing 5 ml of cryoprotective solution, which was then sealed with a heat sealer (Seal Kit 235, Texas Technologies, TX). Subsequently, the samples were transferred to an ice pan before continuing with the freezing method (Fig. 1A and Additional file 1).
Table 1. Description of conditions and reagents used for bioengineered tissue cryopreservation
*See Additional file1
DMSO: dimethyl sulfoxide; hPL: human platelet lysate; ihPL: inactivated human platelet lysate; DMEM: Dulbecco's Modified Eagle´s Medium; FBS: fetal bovine serum; EVA: ethylene-vinyl acetate UPRC: Unidad de Producción y Reprogramación Celular.
Two cryopreservation formulations, termed CeA and CeB, were assessed for cell cryopreservation. The formulations are different to those used in tissue cryopreservation and are both based on ihPL with a reduced DMSO content (5%). For NSC cryopreservation we only assessed CeA, as it provided better results when tested on the other cell types (see results). Detailed information about the cryopreservation solutions and conditions used in each cell type and GMP facility is described in Table 2 and Additional file 1. Cryoprotective solutions were freshly made and kept on ice until use.
Table 2. Description of conditions and reagents used for cell cryopreservation
*See Additional file1
BM-MSCs: bone marrow-derived mesenchymal stromal cells; FBs: human dermal fibroblasts; NSCs: neural stem cells; DMSO: dimethyl sulfoxide; SCB: STEM-CELLBANKER® DMSO FREE-GMP Grade; ihPL: inactivated human platelet lysate; HSA: human serum albumin human; HURS: Hospital Universitario Reina Sofía; UPRC: Unidad de Producción y Reprogramación Celular.
Cell Viability Analysis
We determined the viability of cells embedded in the NFAH before and after cryopreservation using the LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes, Invitrogen, ThermoFisher Scientific). We adapted the manufacturer's protocol by additionally staining cell nuclei with Hoechst solution (Miltenyi Biotec) to aid in cell counting. Cryopreserved NFAHs were thawed by hand (25.5–32ºC) and washed three times with PBS in a Petri dish. Subsequently, cells were incubated with the staining solution for 30 min at room temperature (RT) and then washed three times in PBS. The number of live and dead cells was assessed by fluorescence microscopy, counting three random fields for each NFAH sample.
For cell cryopreservation, we measured viability using trypan blue (Sigma-Aldrich) exclusion before and after thawing BM-MSCs, FBs and NSCs. Cell recovery was calculated according to the following equation: cell recovery post-thaw (%) = (alive cells × 100) / cryopreserved cells. For BM-MSCs and FBs, we additionally evaluated 24 h-recovery as follows: the thawed cells were seeded and cultured at 37ºC and 5% CO2 for 24 hours; cells were then detached with CTS™ TrypLE™ Select Enzyme (A1285901, ThermoFisher Scientific) and counted again. Cell recovery percentage 24-h post-thaw was determined with the following equation: 24-h post-thaw recovery (%) = (alive cells at 24 h × 100) / seeded cells. We performed two cell counts per sample.
Immunofluorescence
NFAHs were examined before and after cryopreservation. NFAHs were fixed in 3.7% buffered formaldehyde for 15 min at RT, and were then dehydrated and embedded in paraffin. For phenotypic analysis, we deparaffinized and rehydrated the NFAH sections and applied heat-induced epitope retrieval with citrate buffer, pH 6 (Alfa Aesar, Haverhill, MA). Subsequently, we permeabilized and blocked the samples with 0.1% Triton X-100 (Sigma-Aldrich) and 5% donkey serum (Sigma-Aldrich) in PBS. Samples were incubated overnight with primary antibodies at 4ºC and then washed twice in PBS and once again in 0.1% Triton X-100 in PBS for 10 min at RT. Finally, we incubated the samples with secondary antibodies for 1 hour at RT (see Additional file 2). All antibodies were diluted in PBS with 2% donkey serum. Nuclei were stained with Hoechst (1:100) and mounted with ProLong™ Gold Antifade Mountant (ThermoFisher Scientific). Analysis was performed using a Nikon TiS microscope (Nikon Instruments, Amsterdam, The Netherlands).
Structural Analysis
NFAH sections were stained with hematoxylin and eosin as described [13, 14] to determine their integrity (interfibrillar space). The preservation degree was determined by the quantification of the spaces produced in the fibrin-agarose fibrillar mesh by ice crystals. Empty areas were quantified with ImageJ software [15] by examining six random fields per NFAH.
Biomechanical Analysis
Evaluation of the biomechanical properties of the NFAHs was performed as described [16]. Briefly, all samples were subjected to tensile tests using an electromechanical material testing instrument (Model 3345-K3327; Instron Ltd., High Wycombe, UK). Samples were sectioned to a regular rectangular shape, oriented with their length along the direction of tension and clamped at each end. A constant distance of 1 cm between the clamps was set. Trials were run at a constant strain rate of 5 mm per min at RT. The following parameters were measured using a 50-N Instron load cell to obtain data for stress–strain curves: Young’s modulus, which characterizes the behavior of elastic material when a force is applied lengthwise, was calculated as the tangent modulus of the initial, linear portion of the stress–strain curve of each experimental run; stress at fracture break, determined by selecting the point of the stress–strain curve where the fracture occurred; and traction/deformation percentage.
Phenotype Assessment
Cells were characterized by flow cytometry before and after cryopreservation. NSCs were characterized one passage after thawing (Additional file 3). Live cells were suspended in PBS and incubated with conjugated antibodies for 30 min at 4ºC in the dark. To test for phenotypic robustness, we first fixed the cells with 3.7% formaldehyde (Sigma-Adrich) for 15 min at RT; they were then blocked and permeabilized with 3% bovine serum albumin (BSA; Sigma-Adrich) and 0.1% Triton X-100 (Sigma-Adrich) in PBS 30 min, RT. Cells were incubated with the antibody for 30 min at 37ºC in the dark. Fluorescence was estimated with a MACS Quant flow cytometer (Miltenyi Biotec) and results were analyzed with MACS Quantify 2.10 software. Isotype controls were run in parallel. The sample size was 10,000 cells per measurement.
Apoptosis And Proliferation
We analyzed apoptosis on FBs before and after cryopreservation using the Annexin-V-FITC Apoptosis Detection Kit (Miltenyi Biotec), as described [17]. As a control, we used 0.5 × 105 cells stained with propidium iodide. Data were analyzed using MacsQuantify 2.10 software. The sample size was 10,000 cells per measurement.
NSCs were seeded and expanded for 1 week (one passage after thawing). Cells were then counted, and the population doubling (PD) was calculated according to the equation: PD = (log y - log x)/ log2, where “y” is the number of cells at the end of the cultivation period and “x” the number of cells at the beginning.
Functional Analysis Of Immunomodulation
Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors collected in heparin tubes. We diluted whole blood 1:1 with Roswell Park Memorial Institute (RPMI) medium (Lonza, Basilea, Sweeden) and layered 2 ml on top of 4 ml of Ficoll solution (Alere Technologies AS, Oslo, Norway) in 15-ml conical tubes. Tubes were centrifuged at 1500 g for 20 min at 20ºC, and the white layer was transferred to a clean tube and washed twice in PBS. PBMCs were stained with 5 µM carboxyfluorescein succinimidyl ester (CFSE; Sigma-Aldrich) for 10 minutes at RT. Cells were then washed twice with PBS and resuspended in RPMI medium.
We seeded 100,000 BM-MSCs per well into a 6-well plate and, 24 hours later, we placed a 0.4 µm-diameter Transwell insert (CellStar, Kaysville UT) over each well and seeded 500,000 PBMCs per insert. PBMCs were activated with 1% phytohemagglutinin (Gibco) 24 h later. The co-culture was maintained for 7 days and PBMC proliferation was assessed by flow cytometry of CFSE expression. To assess T-lymphocyte proliferation, we also stained PBMCs with an antibody to CD3-APC to select the T-lymphocyte population and, again, measured CFSE to detect proliferative T-lymphocytes. Results were analyzed with MACS Quantify 2.10 software.
Stability Analysis
On manufacturing day (t = 0) and after a 3-month storage (t = 3) period at -20ºC, we assessed the stability of ihPL-based solutions allocated for bioengineered tissue cryopreservation. We measured total protein, albumin and immunoglobulin G (IgG) concentrations with a Dimension System platform (Siemens Healthcare, Forchheim, Germany) and pH with a pH-meter (HI 221; HANNA Instruments, Woonsocket, RI). The same parameters were measured for freshly made (t = 0), 12 (t = 12) and 18-month (t = 18) stored solutions (-20ºC) aimed for cell cryopreservation. Furthermore, we evaluated cell solutions to determine an expiry date. With this aim, we cryopreserved FBs (106 cells/ml) with 12- and 18-month stored solutions and with CryoStor® CS10, used as a control.
Statistical analysis
Statistical analysis was conducted with GraphPad Prism, version 8 (GraphPad Software, Inc., La Jolla, CA). To assess normal distribution of data, we used the Shapiro–Wilk test. Levene’s test was used to analyze the assumption of homogeneity of variance. We applied one-way and two-way (for cell density effect assessment) analysis of variance (ANOVA) followed by Tukey’s multiple-comparisons test. Correlation analysis was performed computing the value of the Pearson correlation coefficient, r. Data are expressed as mean and ± standard error of the mean (SEM). P < 0.05 was considered as significant.