Method Optimization of Skin Biopsy-Derived Fibroblast Culture for Reprogramming Into Induced Pluripotent Stem Cells

Abstract

Background:

Fibroblasts can be isolated from skin biopsies using a chemical dissociation, a physical dissociation, or a combination of both techniques. They can be reprogrammed into induced pluripotent stem cells (iPSCs) through the introduction of defined sets of key transcription factors. This study aimed to identify the optimal protocol for skin biopsy dissociation, fibroblast culture, and fibroblast cryopreservation in the scope of reprogramming into iPSCs and in the context of biobank accreditation.

Methods:

First, four dissociation techniques typically used in the laboratory (explant based, enzymatic, and/or mechanical) and two cryopreservation media containing 10% dimethyl sulfoxide, either commercial or homemade, were evaluated in terms of post-thaw recovery, viability, growth curves, and karyotyping analyses of the fibroblasts. Next, the clones reprogrammed from the fibroblasts isolated with the two optimal dissociation methods and cryopreservation media were further assessed by reprogramming quality before cryopreservation and post-thaw pluripotency comparison.

Results:

Fibroblasts isolated from skin biopsies using an explant-based or enzymatic dissociation method showed higher viability, higher proliferative potential, and higher genome stability post-thaw compared to the other dissociation techniques. Fibroblasts obtained by the explant-based dissociation technique showed a slightly higher reprogramming quality. The iPSC reprogrammed from explant-based dissociated fibroblasts showed successful recovery of iPSC clones. No difference between the two cryopreservation media was detected for the tested endpoints, with the exception of a higher visual count of colonies at the end of the reprogramming for the explant-based dissociation method.

Conclusions:

This article presents a formal method optimization for biospecimen processing in the context of accreditation in laboratories and biobanks. We validated skin biopsy-derived fibroblast isolation, culture, and cryopreservation for downstream mRNA reprogramming into iPSCs. The explant-based dissociation technique and homemade medium are selected as optimal to isolate and cryopreserve fibroblasts from skin biopsies in the scope of reprogramming into iPSCs.

Introduction

In this study, we aimed to optimize skin biopsy dissociation and a fibroblast culture method in the scope of reprogramming into induced pluripotent stem cells (iPSCs) and in the context of biobank accreditation. Biospecimen processing method optimization and validation is a normative requirement for biobank accreditation¹ and ensures the method's fitness-for-purpose. We present here the seventh and last article in a series reporting on the optimization and validation of biospecimen processing methods for downstream applications.^2–7

Fibroblasts are a heterogeneous group of cells with diverse functions. They are often defined morphologically as elongated, spindle-shaped cells that adhere and migrate over tissue culture substrates.^8,9 As there are no surface markers exclusively specific for fibroblasts, they are distinguished from other cell lineages by using a combination of markers. Besides their shape, the principal defining characteristic of fibroblasts is their ability to secrete extracellular matrix molecules.¹⁰ Other important functions of fibroblasts include the regulation of epithelial differentiation, regulation of inflammation, and involvement in wound healing.¹⁰

Skin, because of its accessibility, is the best studied tissue with respect to fibroblasts. Several dissociation techniques can be used to isolate fibroblasts from patient-derived skin biopsies. The primary explant technique was the original method to initiate a tissue culture by allowing cells to migrate out from explants adhering to a suitable substrate.^11–13 In contrast, mechanical or enzymatic disaggregation produces a suspension of cells, from which some will adhere to a substrate.¹⁴

Fibroblasts can be reprogrammed into iPSCs through the introduction of defined sets of key transcription factors by various methods, including the mRNA-based method used in this study.¹⁵ iPSC technology has shown potential for in vitro disease modeling, drug discovery, and clinical trials. This potential is increased by combining iPSC technology with genome engineering, which can be used to introduce deletions, insertions of mutations in patient-derived iPSCs.^16,17

The high risk of unreliable experimental results in the area of iPSCs from fibroblasts has been recognized and efforts have been devoted to increase the reliability of the process.¹⁸ Inefficiency of reprogramming is linked to both extrinsic and intrinsic factors¹⁹ and problems of genomic instability have also been reported.²⁰ The therapeutic potential of iPSCs makes understanding factors underlying the reprogramming process of prime importance. The potential impact of the fibroblast isolation and cryopreservation on reprogramming efficiency and genomic stability was assessed in this study.

Materials and Methods

Study design

As shown in Figure 1A, to evaluate the skin dissociation and fibroblast cryopreservation methods, fibroblasts were isolated from skin biopsy triplicates by four dissociation techniques (named, respectively, COLL for enzymatic [collagenase] treatment, PHY for physical separation explant-based treatment, GM for enzymatic and mechanical [GentleMACS Dissociator] treatment, and NS for no separation treatment,) and cryopreserved in two cryopreservation media (the commercial CryoStor^® CS10 named CS10 and the homemade media named DGFD consisting of Dulbecco's modified Eagle's medium [DMEM] containing glutaMAX™ with 20% fetal bovine serum [FBS] and 10% dimethyl sulfoxide [DMSO]).

FIG. 1.

Schematic representation of the study design. Four dissociation techniques (namely COLL, PHY, GM, and NS) for isolation of fibroblasts from skin biopsies and two cryopreservation media (namely CS10 and DGFD) for fibroblast cryopreservation were tested (A). Reprogramming assessment performed immediately after the reprogramming (without a cryopreservation step) and post-thaw for the two optimal dissociation techniques (COLL and PHY) and two cryopreservation media (CS10 and DGFD) (B).

To evaluate the optimal skin dissociation and fibroblast cryopreservation method, a comparison of post-thaw passage P8 fibroblasts for recovery, viability, growth curve, and karyotyping was performed.

To evaluate the fitness-for-purpose of the optimal dissociation and cryopreservation method in terms of reprogramming suitability, we reprogrammed post-thaw passage P3 fibroblasts isolated with the COLL and PHY dissociation techniques and cryopreserved in CS10 and DGFD (Fig. 1B). Comparison of iPSCs was performed at two different time points: immediately after expansion (without a cryopreservation step) and post-thaw after a cryopreservation step (Fig. 1B). The iPSCs of passage P4 to P6 were used for immediate characterization, while iPSCs of passage P8 were used for post-thaw characterization. Immediate assessment was performed by comparison of a visual count of colonies at the endpoint of reprogramming and by reprogramming quality. For the purpose of this study, the reprogramming quality was defined as the ratio of number of positive clones to the number of candidate clones. Candidate clones are clones that survived without differentiating during the expansion phase and showed all phenotypic characteristics of iPSCs. The positive clones are candidate clones that were further positively characterized by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (Fig. 1B). For post-thaw clones, comparison of pluripotency, as assessed by qRT-PCR and immunofluorescence (IF), and of trilineage differentiation, as assessed by IF, was performed. The complete dataset used in this study is available here: doi.org/10.17881/255d-4a98.

Skin sample

One skin abdominoplasty was used for this study. The skin sample was provided by the CRB-CHU Amiens, Biobanque de Picardie (Biobanking Resources Impact Factor: BB-0033-00017) under approval from the Ministère de la Recherche (No. AC-2013-1827). The skin sample was removed as part of an esthetic surgical procedure and immediately shipped to IBBL at room temperature (RT), immersed in DMEM containing glutaMAX (Cat No. 21885; Gibco) supplemented with 1% Penicillin/Streptomycin (Pen/Strep, Cat No. 15070; Gibco). Upon reception, the next day, 12 skin biopsies were performed using biopsy punches [Cat No. 05.SF004.11/08(201); Stiefel] and placed in 2 mL tubes (Cat No. 72.694.007; Sarstedt) containing 1.8 mL of the above transport medium.

Fibroblast isolation by four dissociation techniques (COLL, PHY, GM, and NS) and cryopreservation in two cryopreservation media (CS10 and DGFD)

As shown in Figure 1A, four dissociation techniques named COLL, PHY, GM, and NS were tested. For the COLL technique, each skin biopsy was disrupted with a blade (Cat No. 0311; Swann-Morton) in 2 mL of filter-sterilized (Cat No. 130-095-823; Miltenyi Biotec) 10 × collagenase solution (1 g of Collagenase Type II lyophilized [Cat No. 17101; Gibco] diluted in 12.5 mL of culture media) and incubated for 45 minutes at 37°C in a 5% CO₂ incubator.²¹ The digested tissue was centrifuged at 200 g for 5 minutes at RT and washed twice with 3 mL of culture medium. Each pellet was resuspended in 5 mL culture medium and dispensed in a culture flask (Cat No. 690-175; Greiner) for culture. Following the same protocol, dissociation of skin biopsies in triplicate was also tested with a 1 × collagenase solution (100 mg of Collagenase Type II lyophilized diluted in 12.5 mL of culture media). For the PHY technique, each skin biopsy was cut into seven explants with a blade and distributed with the dermis facing down the culture flask covered in FBS (Cat No. 16140-063; Gibco). After an incubation of 10 minutes at RT, 5 mL of culture medium was added to each flask for culture.²² For the GM technique, the whole skin dissociation kit (Cat No. 130-101-540; Miltenyi Biotec) was used following the manufacturer's instructions, with the following adaptations: each 5 mm diameter biopsy was cut in two, no enzyme P was used, and an overnight incubation in a 37°C water bath was done. GentleMACS C tubes (Cat No. 130-093-237; Miltenyi Biotec) on the GentleMACS Dissociator (Miltenyi Biotec) were used. For the NS technique, each skin biopsy was sheared off with a scalpel (cut into the shape of a star) and placed with the dermis facing down in a FBS-covered culture flask. After an incubation of 10 minutes at RT, 5 mL of culture medium was added to each flask. The culture medium consisted of DMEM supplemented with 10% FBS and 1% Pen/Strep (Cat No. 15140; Gibco).

After dissociation, fibroblast cell culture was performed under standardized conditions in culture media at 37°C in a 5% CO2 incubator. At 70–90% confluency, the fibroblasts were rinsed with 1 × Dulbecco's phosphate-buffered saline (D-PBS) (Cat No. 14190; Invitrogen), then trypsinized with a Trypsin/EDTA solution (Cat No. CC-5012; Lonza), incubated at 37°C for 5–10 minutes, and centrifuged at 400 g for 10 minutes at RT for subsequent subculture or cryopreservation. Fibroblasts were cryopreserved using either a controlled rate freezer (Sylab), lowering the temperature from 4°C to −80°C at 1°C per minute, or a Mr. Frosty™ (Thermo Scientific™) in two different cryopreservation media referenced as CS10 for CryoStor CS10 (Cat No. 210102; Biolife Solutions) and DGFD for DMEM containing glutaMAX +20% FBS +10% DMSO (Cat No. D2650; Sigma). The cryopreserved cells were stored in liquid nitrogen (LN). The concentration of total cryopreserved fibroblasts was 1–2 × 10⁶ cells/mL of cryoprotectant. Absence of mycoplasma contamination before cryopreservation was confirmed using MycoAlert™ PLUS mycoplasma detection kit (Cat No. LT07-703; Lonza).

To thaw fibroblast samples, cryovials were incubated in a 37°C water bath until ice crystals disappeared. The cells were gently transferred into a tube containing culture media and immediately centrifuged at 400 g for 10 minutes at RT before culture.

Fibroblast characterization

Fibroblast recovery and viability were assessed using a Cellometer Auto 2000 Cell Profiler (Nexcelom Bioscience) equipped with Cellometer Auto2000 v1.2.7.4 software, using Cellometer AOPI staining solution in PBS (Cat No. CS2-0106; Nexcelom Bioscience), Cellometer Cell counting chambers (Cat No. SD100; Nexcelom Bioscience), and an in-house validated fibroblast counting assay. Immediate recovery post-thaw was calculated using the following formula: recovery (%) = [(yield of viable cells after thawing)/(yield of cryopreserved viable cells)*100]. The cell yield fold change on day 3 was calculated as Y_Day3/Y_Day0, where Y stands for yield of viable cells.

Cell apoptosis was assessed by flow cytometry analysis using the FITC Annexin V Apoptosis Detection Kit 1 (Cat No. 556547; BD Pharmingen) according to the manufacturer's instructions on a BD FACSVerse with FACSuite v1.0.4.2650 software. In this kit, single Annexin V-stained cells are reported as “early apoptotic,” and double Annexin V/propidium iodide (PI)-stained cells are reported as “late apoptotic” cells.

Growth curves were calculated from confluence measurements performed with a Cytonote lens-free video microscope (Iprasense). Each cell sample was seeded (day-2) in three different 24-well plates (Cat No. 142475; Thermo Fisher). Confluence measurements were performed on day 0, 2, 4, and 6 and normalized to the day 0 confluence measurement. The slope of each linear trendline was measured for the mean confluence of the three plates.

Fibroblast karyotyping was performed by two techniques: Giemsa banding (G-banding) and molecular karyotyping. Fresh fibroblasts of passage P8 were G-banded with trypsin to a resolution of ∼400 bands per cell.²³ Chromosome analysis was performed using a standard light microscope and a minimum of 20 metaphases per sample were karyotyped.²⁴ Genomic DNA from technical replicates of post-thaw passage P10 fibroblasts cryopreserved in CS10 was isolated using DNeasy Blood and Tissue kit (Cat No. 69504; Qiagen). Samples were analyzed at the Life & Brain genomics facility from Bonn University, using Illumina iScan technology (Illumina) and the HumanOmniExpressExome-8 Beadchip v1.

Reprogramming

To generate iPSCs, fibroblasts (1.5 × 10⁵ cells) were reprogrammed using the Ribojuice™ mRNA Transfection kit (Cat No. TR-1013; Millipore) and Simplicon™ RNA Reprogramming kit (Cat No. SCR550; Millipore) following the manufacturer's instructions. Cells were cultured in iPSC medium consisting of Essential 8™ Medium (Cat No. A1517001; Thermo Fischer) supplemented with 1% Pen/Strep. Emerging iPSC colonies with clear iPSC morphology were manually picked and seeded onto Matrigel (Cat No. 354277; Corning)-coated plates. iPSC medium was exchanged daily. Expansion and cryopreservation were performed by mechanical passaging with 0.5 mM EDTA (UltraPure™ 0.5 M EDTA [Cat No. 15575020; ThermoFisher Scientific] diluted in 1 × D-PBS). iPSC clones were cryopreserved using either a controlled rate freezer, lowering the temperature from 4°C to −80°C at 1°C per minute, or a Mr. Frosty in CS10. The cryopreserved cells were transferred and stored in LN.

Cryovials with iPSCs were thawed in a 37°C water bath until only small ice crystals remained and transferred dropwise to a conical tube containing prewarmed medium. The cell suspension was centrifuged at 200 g, 5 minutes, at RT before subsequent subculture.

iPSC characterization

Immediate iPSC characterization was performed by pluripotency assessment by qRT-PCR, while post-thaw characterization included both pluripotency assessment by qRT-PCR and, IF, and trilineage differentiation assessment by IF.

For qRT-PCR characterization, RNA was extracted from QIAzol-lysed iPSCs using the miRNeasy Mini Kit (Cat No. 217004; Qiagen) following the manufacturer's instructions. RNA yields were analyzed with a Synergy MX (BioTek) spectrophotometer. For each sample, 1 μm of total RNA was transcribed using the High-Capacity Reverse Transcription Kit (Cat No. 4374966; Life Technologies) in a total volume of 20 μL on a C1000 PCR thermal cycler (Bio-Rad, Belgium). The cDNA was diluted 7.5 times to a total volume of 150 μL and mRNA samples were quantified using commercial TaqMan assays (Part No. 4331182; Life Technologies) according to the manufacturer's instructions using commercial probes (hEID2 [assay ID: Hs00541978_s1], hSOX2 [assay ID: Hs01053049_s1], hPOU5F1 [assay ID: Hs04260367_gH], and hNanog [assay ID: Hs02387400_g1]; Life Technologies). qPCR reactions were run in triplicate using 5 μL diluted cDNA in a total volume of 20 μL on the ABI 7500 Fast Real-Time PCR System (Life Technologies), and they were analyzed using 7500 System SDS software (v1.4), with results scored as cycle thresholds (Cts). The relative quantification (RQ), calculated using the formula RQ = 2^−ddCt, gives the relative expression level of genes calculated by normalizing their Ct values with that of the housekeeping gene hEID2²⁵ and calibrated to a cDNA calibrator (Cat No. 740000; Agilent Technologies). The pluripotency RQ range of each target was defined using a pluripotent reference material and pluripotent samples characterized by IF and by the TaqMan^® hPSC Scorecard Panel kit (Cat No. A15871; Thermo Fisher Scientific). A clone was described as positive when all RQ results of the three targets selected were within the predefined range.

For IF characterization, cells were fixed for 15 minutes at RT with 4% paraformaldehyde (Cat No. 19943; Affymetrix) in 1 × D-PBS and washed thrice with 1 × D-PBS for 5 minutes at RT. Permeabilization was performed using 0.5% Triton-X100 (Cat No. X100; Sigma) in 1 × D-PBS for 15 minutes at RT and then washed thrice. Blocking was performed for 1 hour at RT using 10% FBS (Cat No. 16140; Gibco) in 1 × D-PBS. Incubation with the corresponding primary antibodies at the required concentrations was done overnight at 4°C in blocking buffer. After three washing steps, incubation with secondary antibodies (1:1000, Cat No. A21202 and A10042; Invitrogen), together with a nuclei counterstain Hoechst 33342 (1:10,000, Cat No. H3570; ThermoFisher Scientific), was done for 1 hour at RT in blocking buffer. After three washing steps, coverslips were mounted with Fluoromount-G mounting medium (Cat No. 0100-01; Southern Biotech) and imaged on an Axio Observer.Z1 microscope (Zeiss).

The targets used for pluripotency staining were SSEA4 (1:25, Cat No. MAB4304; Millipore), Nanog (1:100, Cat No. AB5731; Millipore), TRA-1-60 (1:25, Cat No. MAB4360; Millipore), Oct4 (1:600, Cat No. ab19857; Abcam), TRA-1-81 (1:25, Cat No. MAB4381; Millipore), and SOX2 (1:200, Cat No. ab97959; Abcam).

For trilineage differentiation, cells were differentiated in their specific lineage (endoderm, mesoderm, and ectoderm) by targeted differentiation following protocols established previously.^26–28 The IF characterization was performed as described above. The targets used were SOX17 (1:13, Cat No. IC19241G; R&D Systems) for endoderm lineage, alpha smooth muscle actin antibody (1:100, Cat No. ab5694; Abcam) for mesoderm lineage, and SOX1 (1:100, Cat No. AF3369; R&D Systems) and β-tubulin (TUBB3) (1:10, Cat No. 560338; BD Biosciences) for ectoderm lineage.

Statistical analysis

For the optimal dissociation technique, a one-way ANOVA was used for analysis of data before cryopreservation. A two-way ANOVA was used for post-thaw data analysis. When treatments were significantly different, a Holm-Sidak test for pairwise comparisons was performed. For the optimal cryopreservation media, a two-tailed t-test was used. All analyses were performed using SigmaPlot version 12.5 with significance level p < 0.05. All graphs were created in Excel version 2003.

Results

Optimal skin dissociation and fibroblast cryopreservation method

Our objective was to identify the optimal method for skin biopsy dissociation, fibroblast culture, and cryopreservation. For this purpose, we compared the performance characteristics of four dissociation techniques (COLL, PHY, GM, and NS) in combination with two cryopreservation media (namely CS10 and DGFD), as described in the Materials and Methods section.

Cell recovery

As shown in Figure 2A, post-thaw cell recovery, measured by AOPI staining intensity with the Cellometer, ranged between 73.3% and 103.0%. There was no significant difference in recovery among the dissociation techniques or cryopreservation media tested. The cell yield fold change at 3 days post-thaw compared to the yield of viable cells at day 0 ranged between 1.3 and 2.1 (Fig. 2B). There was no significant difference in cell yield fold change among the dissociation techniques and cryopreservation media combinations.

FIG. 2.

Performance characteristics of fibroblasts obtained with the four dissociation techniques (COLL, PHY, GM, and NS) and two cryopreservation media (CS10 and DGFD) (n = 3). The mean post-thaw recovery (A), 3 days post-thaw fold change (B), post-thaw viability (%) (C), 3 days post-thaw viability (%) (D), post-thaw apoptosis levels (%) (E), and growth curves (F) are shown. Error bars are one standard deviation and statistically significant differences (p < 0.05) are denoted by *.

Cellometer-based characterization

Concerning viability, measured by AOPI staining intensity with the Cellometer, there was no significant difference among the different dissociation techniques before cryopreservation, with viability ranging between 95.4% and 98.2%. For post-thaw viability, measured by AOPI staining intensity, the fibroblasts isolated with the GM dissociation technique showed significantly lower viability (average 69.4%) compared to those isolated with the COLL, PHY, or NS techniques, which had an average 89.8%, 91.7%, and 89.3% viability, respectively (p < 0.05), while no significant difference was detected between the two cryopreservation media (Fig. 2C). At 3 days post-thaw, the GM-isolated fibroblast viability, measured by AOPI staining intensity, remained significantly lower (average 84.8%) compared to those isolated with the COLL technique (average 95.0%, p = 0.047), but not compared to those isolated with the PHY or NS techniques, which had an average 86.8% and 89.7% viability, respectively, while no significant difference was detected between the two cryopreservation media (Fig. 2D).

Flow cytometry-based characterization

Following post-thaw FACSVerse measurement of Annexin V- and PI-stained cells, the fibroblasts isolated with the GM dissociation technique showed significantly lower viable cell levels (77.1%) than those isolated with the COLL (93.6%), PHY (87.9%), and NS (89.9%) techniques (p < 0.05), while no significant difference between the cryopreservation media was found (Fig. 2E).

Growth rates

The post-thaw growth curve slope values, measured by confluence measurements with a Cytonote, showed that the fibroblasts isolated with the COLL dissociation technique proliferated significantly quicker (slope = 1.9) than those isolated with the PHY (slope = 1.2), GM (slope = 0.8), and NS (slope = 0.7) techniques (p < 0.05). There was no significant difference of fibroblast growth rate between the two cryopreservation media (Fig. 2F).

Karyotyping

Karyotyping of the fibroblasts was performed by G-banding and by molecular karyotyping. A combination of both methods is required as array-based methods cannot accurately detect balanced inversions and translocations.²⁹ For the G-banding analyses, 90% of the mitotic fibroblasts produced by the COLL dissociation technique presented no abnormalities or nonclonal abnormalities, while 10% presented a modification of chromosomal structure of translocation type t(X;8). Only 76% of the mitotic fibroblasts produced by the GM dissociation technique presented no abnormalities or nonclonal abnormalities, while 14% presented a structural chromosomal modification of translocation type t(3;6), and 10% presented three independent nonclonal structural abnormalities [del(5)(p), dup(7)(p) and der(17)]. One hundred percent of the mitotic fibroblasts produced by the PHY dissociation technique presented no abnormalities or nonclonal abnormalities. Ninety percent of the mitotic fibroblasts produced by the NS dissociation technique presented no abnormalities or nonclonal abnormalities, while 10% of them presented a structural anomaly with unidentified supplementary materials on the short arm of chromosome 13.

Molecular karyotype assessment performed with an Illumina Bead Array—Illumina iScan system revealed no major abnormality for the fibroblasts isolated with the dissociation techniques COLL, PHY, and NS. For the GM technique, no large chromosomal aberrations were reported for the first biological replicate, but a 27 Mbp large deletion on the long arm of the chromosome 5 was reported for the second biological replicate and a 27 Mbp large deletion mosaic (75%) on the long arm of the chromosome 5 was reported for the third biological replicate.

The COLL and PHY dissociation techniques were selected as optimal, as they were associated with higher fibroblast recovery, viability, growth, and normal karyotypes, compared to the GM and NS dissociation techniques. No difference in any of the measured endpoints was detected between the cryopreservation media CS10 and DGFD.

Reprogramming assessment immediately after expansion (without a cryopreservation step) of fibroblasts produced with the COLL and PHY dissociation techniques

Our objective was to verify and assess the fitness-for-purpose of fibroblasts isolated with the COLL and PHY dissociation techniques for reprogramming into iPSCs. Comparison of the cryopreservation media CS10 and DGFD was also performed in the scope of reprogramming suitability.

Fibroblasts produced with the COLL dissociation technique

For the COLL dissociation technique as shown in Table 1, no significant difference was found between the cryopreservation media for the numbers of visually counted colonies, observed at the end of reprogramming on day 36, for fibroblasts frozen in the two cryopreservation media (Table 1).

Table 1.

Reprogramming Assessment Immediately After Expansion (Without a Cryopreservation Step) and Post-Thaw for the Two Optimal Dissociation Techniques (COLL and PHY) and Cryopreservation Media (CS10 and DGFD) (n = 3)

Time point assessment	Performance characteristic measured	Measurement device	COLL		PHY
Time point assessment	Performance characteristic measured	Measurement device	CS10	DGFD	CS10	DGFD
Assessment immediately after expansion (without a cryopreservation step)	Visual count of colonies at the end of the reprogramming	—	112 ± 11	91 ± 10	28 ± 3	42 ± 3
	Number of colonies picked up	—	18 ± 0	18 ± 0	12 ± 0	18 ± 6
	Number of candidate clones cryopreserved	—	8 ± 1	8 ± 2	7 ± 1	6 ± 1
	Reprogramming efficiency (%)	Fast Real-Time PCR AB7500	32 ± 6	19 ± 5	36 ± 23	39 ± 18
Post-thaw assessment	Ratio number of clones IF positive for pluripotency/number of clones thawed	Axio Observer.Z1 microscope	3/3	3/3	3/3	3/3
	Ratio number of clones with gene expression in pluripotency range/number of clones thawed	Fast Real-Time PCR AB7500	2/3	0/3	1/3	3/3
	Ratio number of clones IF positive for trilineage differentiation/number of clones thawed	Axio Observer.Z1 microscope	3/3	3/3	3/3	3/3

IF, immunofluorescence; PCR, polymerase chain reaction.

A total of 108 colonies were picked for the 3 technical replicates and the 2 cryopreservation media tested. Of these, 49 iPSC clones survived the expansion phase with 24 clones reprogrammed from CS10-cryopreserved fibroblasts and 25 from DGFD-cryopreserved fibroblasts. Ultimately, 38 iPSC clones were defined as candidates (18 from CS10-cryopreserved fibroblasts and 20 from DGFD-cryopreserved fibroblasts). No significant difference was detected between the two cryopreservation media for the reprogramming quality (ratio of the number of positive clones to the number of candidate clones) (p = 0.053) (Table 1).

Fibroblasts produced with the PHY dissociation technique

For the PHYdissociation technique as shown in Table 1, a significantly higher number of colonies were measured at the end of the reprogramming on day 46 for the samples reprogrammed from DGFD-cryopreserved fibroblasts (p = 0.005) (Table 1).

A total of 90 colonies were picked for the 3 technical replicates and the 2 cryopreservation media tested. Of these, 40 iPSC clones survived the expansion phase with 21 clones that were reprogrammed from CS10-cryopreserved fibroblasts and 19 reprogrammed from DGFD-cryopreserved fibroblasts. Ultimately, 34 iPSC clones were defined as candidates (17 from CS10-cryopreserved fibroblasts and 15 for DGFD-cryopreserved fibroblasts). No significant difference was detected between the two cryopreservation media for the reprogramming quality (p = 0.843) (Table 1).

In conclusion, our results demonstrate that fibroblasts obtained with both the COLL and PHY skin dissociation techniques are fit-for-purpose for iPSC reprogramming, with fibroblasts obtained with the PHY dissociation technique showing a slightly higher reprogramming quality. Concerning the two cryopreservation media, no significant difference was found for the different endpoints, with one exception. A higher number of colonies measured at the endpoint of analysis were obtained for the samples reprogrammed from DGFD-cryopreserved fibroblasts produced with the PHY dissociation technique.

Post-thaw pluripotency assessment of clones reprogrammed from fibroblasts produced with the COLL and PHY dissociation techniques

Finally, we verified the post-thaw pluripotency of clones reprogrammed from the fibroblasts that had been initially produced with the COLL and PHY dissociation techniques and cryopreserved in the two cryopreservation media CS10 and DGFD (Fig. 1B).

Six COLL-positive clones were thawed: one clone reprogrammed from fibroblasts cryopreserved in CS10 (“COLL-CS10”) obtained from each replicate (S1, S2, and S3) and one clone reprogrammed from fibroblasts cryopreserved in DGFD (“COLL-DGFD”) from each replicate (S1, S2, and S3). All clones survived the expansion phase and were characterized for pluripotency by qRT-PCR, IF, and for trilineage differentiation by IF. All clones expressed typical pluripotency IF signals for all six targets Nanog, SSEA4, Oct4, TRA-1-60, TRA-1-81, and SOX2 (Fig. 3A) and typical IF signals for each lineage differentiation (SOX17 for endoderm, α-smooth muscle actin for mesoderm, and SOX1 and TUBB3 for ectoderm) (Fig. 3B). Two of the six clones had the relative gene expression of all the target genes within the expected pluripotency status ranges (Fig. 4). The post-thaw recovery of cryopreserved iPSC was therefore low, but successful for “COLL-CS10.”

FIG. 3.

Representative images of pluripotency and trilineage assessment of post-thaw iPSC clones, reprogrammed from fibroblasts, initially isolated with the COLL or PHY technique and cryopreserved in CS10 or DGFD media, by immunofluorescence staining. Image magnification is 20 × . For pluripotency assessment, the six targets tested were Nanog, SSEA4, Oct4, TRA-1-60, TRA-1-81, and SOX2 (A). For trilineage analysis, the targets tested were SOX17 for endoderm lineage, alpha-smooth muscle actin for mesoderm lineage, and SOX1 and TUBB3 for ectoderm lineage (B).

FIG. 4.

RQ results of target gene expression levels in post-thaw iPSC clones, reprogrammed from fibroblasts, initially isolated with the COLL or PHY techniques, and cryopreserved in CS10 or DGFD media. Target genes are hSOX2, hPOU5F1, and hNANOG. The expected RQ range for pluripotency of each target is indicated by the horizontal lines. Error bars are one standard deviation. RQ, relative quantification.

Six PHY-positive clones were thawed: a “PHY-CS10” and a “PHY-DGFD” clone for each replicate (S1, S2, and S3). All clones survived the expansion phase and were fully characterized. All clones expressed all typical pluripotency IF signals for the six targets Nanog, SSEA4, Oct4, TRA-1-60, TRA-1-81, and SOX2 (Fig. 3A), and typical IF signals for each lineage differentiation (SOX17 for endoderm, α-smooth muscle actin for mesoderm, and SOX1 and TUBB3 for ectoderm) (Fig. 3B). Four clones had the relative gene expression levels of all three target genes within the predefined ranges (Fig. 4). The post-thaw recovery of cryopreserved iPSCs was successful.

To conclude, the post-thaw recovery of cryopreserved iPSC initially dissociated with the COLL method was low, but successful for fibroblasts cryopreserved with CS10. For iPSCs reprogrammed from PHY-isolated fibroblasts, the fibroblast cryopreservation method using both cryopreservation media showed successful post-thawing recovery of fully pluripotent iPSC clones with the highest success rate with DGFD-cryopreserved fibroblasts.

Discussion

Good Cell Culture Practice (GCCP) for primary cell culture requires sufficient understanding of the in vitro system and the critical factors that can affect the performance characteristics of the cellular end products.³⁰ In our study, primary cell culture of fibroblasts was initiated from 12 skin biopsies, excised from one skin surgical abdominoplasty, to eliminate potential bias resulting from genetic variations between different individuals. Fibroblasts isolated from different anatomical regions exhibit topological variation, as demonstrated by early work on the heterogeneity of fibroblasts,³¹ and by more recent studies using microarray technology.^32–34 Topological variation was shown to be the principal source of variation in the genomic expression profiles in a comparison of cultured cells that had variable donor sources, passage numbers, and presence or absence of serum in the culture medium.³³ Fibroblasts derived from different anatomical sites have shown differences in their reprogramming efficiencies.³⁵ In another study, donor age and passage number of fibroblasts have been shown to impact their reprogramming efficiency.³⁶ To analyze the impact of skin dissociation techniques and the cryopreservation media, the above cited variables were fixed throughout the study.

Different easily applicable dissociation techniques were selected to ensure feasibility. Explant-based techniques will select for fibroblasts on the basis of cell migration capacity, while enzymatic or mechanical dissociation-based techniques will select for protease- and mechanical stress-resistant cells.³⁷ For the enzymatic dissociation, collagenase was selected as the treatment of reference, instead of other enzymatic options, since the skin extracellular matrix often contains collagen, particularly in the connective tissue and muscle.³⁷ The Collagenase Type II used was isolated from Clostridium histolyticum. A crude preparation was used as this is often more successful than purified enzyme preparations.³⁷ Dissociation of skin biopsies in triplicate was tested with collagenase 1 × and 10 ×, and no difference in the viable cell yield, viability, or growth curves was observed after 7 days of culture (data not shown). The concentration of collagenase used in this study was the 10 × . At first passage, the highest mean viable cell yield was found for the COLL-dissociated fibroblasts, although no significant difference between the yield of viable cells and viability was detected between the dissociation techniques. The collagenase-treated fibroblasts exhibited increased proliferative capacity when compared to the fibroblasts produced with other dissociation techniques. This increased proliferative capacity was verified at all 10 passages performed.

Two different techniques were used to detect fibroblast genomic instability. The G-banding conventional method was used to detect numerical (aneuploidy and polyploidy) or large structural chromosomal modifications. The Illumina Bead Array provided higher resolution and permitted study of copy number variations of the whole genome at kilobase resolution. A combination of both methods is, however, required as array-based methods cannot accurately detect balanced inversions and translocations.²⁹ Combining the results from the two techniques, a higher structural instability was detected for the fibroblasts isolated with the GM skin dissociation technique, with the chromosome 5 found to be particularly unstable.

Two cryopreservation media, with 10% DMSO as the cryoprotective agent, were used to cryopreserve the isolated fibroblasts. DMSO is an intracellular cryoprotectant that reduces ice crystal-associated cell damage by displacing water from within the cells during freezing. The product CryoStor contains dextran-40, sodium, potassium, calcium, magnesium, phosphate, HEPES, lactobionate, sucrose, mannitol, glucose, adenosine, and glutathione.³⁸ This serum-free standardized medium eliminates batch-to-batch variations of homemade cryopreservation media. Previous publications indicate that CryoStor significantly improves post-thaw cell survival.^38,39 In our study, no difference between the homemade DGFD cryopreservation medium and the CryoStor CS10 was observed for the post-thaw recovery, viability, apoptosis levels, and growth of fibroblasts.

As genetic and epigenetic heterogeneity in human iPSCs are attributable to the method by which they are initially produced, one specific reprogramming method was applied throughout this study.⁴⁰ For higher similarity between iPSCs and embryonic stem cells, methods requiring transfection are preferred to those requiring electroporation, while viral methods have shown highest genetic similarity compared to transfection-based reprogramming methods.⁴⁰ The integration-free mRNA reprogramming method was used in this study.⁴¹ A single population of fibroblasts was used to eliminate interindividual genetic variation.

Cryopreservation of the iPSCs should maintain their critical quality attributes. Overall, the post-thaw recovery of pluripotent clones, cryopreserved as colonies, was successful. Another possibility is to cryopreserve iPSCs as single-cell suspensions. Although single cells are not recommended for iPSC applications, the use of Rho-associated kinase (ROCK) inhibitor allows good recovery from single-cell formulations.⁴²

Potential confounding processing factors were tightly controlled in this study. The same culture, reprogramming, and analysis protocols were performed throughout the study. As recommended by GCCP, all references and protocols are detailed. Absence of mycoplasma contamination was confirmed throughout the whole culture process. Several limitations of the study can be noted. For the purpose of this study, the reprogramming quality was defined as the ratio of number of positive clones to the number of candidate clones, which is different from the traditionally reprogramming efficiency definition based on the number of iPSC colonies generated, divided by the number of input cells. For biobanking purposes, time-efficient acquisition of iPSCs instead of colonies was defined as the main topic of interest. As such, the reprogramming quality definition used in this study takes into account the reprogramming and the expansion efficiency. No quantitative comparison can be made between the traditional reprogramming efficiency of fibroblasts produced with the COLL or PHY technique, as the same days were not applied for the endpoint of analysis (day 36 and 46). Skin biopsies were obtained only from one individual. This limitation was required to eliminate potential bias resulting from genetic variations between different individuals. No comparison of the genome stability between the iPSC clones obtained with the different dissociation techniques was performed. The results and conclusions of our study are limited to the specific protocols that were applied during processing of the fibroblasts. It cannot be excluded that different enzyme concentrations or types, different incubation times, or different mechanical forces applied during homogenization might have led to different results.

As a conclusion, the dissociation technique used to isolate fibroblasts from skin biopsies in the scope of reprogramming into iPSCs is a critical factor, which should be optimized. In this study, combinations of four skin dissociation techniques for fibroblast isolation and two cryopreservation media were tested. Following thorough assessment of fibroblast quality, the PHY dissociation technique was considered optimal. Fibroblasts obtained with this method were further shown to be fit-for-purpose for reprogramming efficiently into iPSCs. Concerning the cryopreservation media, both media were considered fit-for-purpose for fibroblast cryopreservation in the scope of reprogramming into iPSCs. Based on the data, taking into account the above limitations, the PHY dissociation technique and homemade media DGFD are selected as optimal to isolate and cryopreserve fibroblasts from skin biopsies in the scope of reprogramming into iPSCs.

Footnotes

Acknowledgments

We thank Inga Werthschulte for the reprogramming training and tips. We acknowledge the contribution from Dr. Geneviève Lefort, Marc Tapp, and Nicolas Perosino of Laboratoire National de Santé (LNS) who performed the G-banding analyses. We gratefully thank Wim Ammerlaan for the flow cytometry support. We thank Yves Van Fraeyenhoven for the image help.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

Funding for this research was provided by internal resources of the Integrated BioBank of Luxembourg. No external funding was utilized.

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