Heregulin Activity Assays for Residual Testing of Cell Therapy Products

Cell Culture Materials, Antibodies, and Reagents

Cell culture-grade water, high-glucose Dulbecco’s Modified Eagle’s medium (DMEM), DMEM/F12 (1:1) with L-glutamine, 15 mM HEPES and without phenol red (Invitrogen, Cat 11039), Hanks Balanced Salt Solution (HBSS, calcium- and magnesium-free), Dulbecco’s Phosphate-Buffered Saline (DPBS), 0.5% trypsin-EDTA, 10,000 U/mL penicillin-streptomycin and 50 mg/mL gentamycin were obtained from ThermoFisher (Waltham, MA). Fetal bovine serum (FBS) was obtained from HyCloneTM-GE Healthcare (Logan, UT) and decomplemented in-house. Forskolin, poly-L-lysine (PLL), mouse laminin (1 mg/mL) and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich (St. Louis, MO). The EGFR/ErbB2 inhibitor 4557 W (CAS 179248–61-4) was from Calbiochem-Novabiochem Corp, La Jolla, CA. The following primary antibodies used for immunostaining were obtained from DAKO (Carpinteria, CA): anti-S100β (rabbit polyclonal, Cat # Z0311) and anti-GFAP (glial fibrillar acidic protein rabbit polyclonal, Cat # Z0334). The following antibodies used for Western blot were obtained from Cell Signaling Technology: anti-phosphorylated (Ser217/221)-MEK (cat. # 9121), and anti-phosphorylated-tyrosine (Cat. # 9411). Alexa Fluor™ 488 and 546-conjugated antibodies in the form of goat anti-rabbit IgG (Cat # A-11035), and goat anti-mouse IgG (Cat # A-11001) were acquired from ThermoFisher. Thy1.1 antibodies were prepared in-house from hybridoma cell cultures (ATCC, Manassas, VA), according to our published protocols [14]. All disposable plasticware and cell culture dishes were obtained from Corning. Recombinant human heregulin-β1177–244 (HRG1-B1, Preprotech, cat # G-100-03) was prepared in sterile water and stored in aliquots at − 80 °C, as recommended by the manufacturer. Other reagents and equipment are described in the appropriate sections.

Preparation and Characterization of Stock Cultures of Rat SCs

All procedures involving animals were approved by the University of Miami Animal Care and Use Committee. Rat SC cultures were established from adult (3-month-old) female Fisher rat sciatic nerve tissue, as previously described [11, 12]. Briefly, the sciatic nerve tissue was cut into small segments and allowed to degenerate in vitro by incubation for 2–3 weeks in DMEM medium supplemented with 10% heat-inactivated FBS (DMEM-10% FBS). Degenerated nerve explants were dissociated with a mixture of 0.25% dispase and 0.05% collagenase, and the resulting cell suspensions were plated on PLL-coated 10-cm dishes in DMEM-10% FBS. Contaminating fibroblasts were removed by a complement reaction using Thy 1.1 antibodies. The resulting purified SCs were expanded in DMEM-10% FBS medium supplemented with 2 μM forskolin, 20 μg/ml bovine pituitary extract (Biomedical Tech., Stoughton, MA), and 10 nM β1-heregulin (Genentech). SCs were expanded in the aforementioned medium up to passage-2 before cryogenic storage. Experiments were carried out using cryopreserved cells prepared in RecoveryTM medium or dimethyl sulfoxide (DMSO) and FBS at a ratio of 1:9. Cryopreservation was performed to create large enough stocks of rat SCs from the same batch for use in all heregulin activity assays, thus minimizing lot-to-lot variability in the kinase activation measurements. Adult rat SC cultures consisted of > 98% SCs based on immunostaining with the SC markers S100B or GFAP, and the fibroblast marker Thy1.1 [15]. Cultures obtained by this method were analyzed by RNAseq to confirm the purity and identity of the cells. These cultures expressed ErbB2 and ErbB3 mRNAs but lacked NRG isoforms, EGFR and ErbB4. More information on the RNAseq analysis of rat SC cultures can be found in our prior publication [7].

Preparation and Storage of in-Process Residual Testing Samples, Positive and Negative Controls

Samples from the culture medium of hSCs were obtained from 3 independent process qualification runs carried out in a facility operating under current Good Manufacturing Practices (cGMP). Several manufacturing process steps and in-process controls were employed to ensure that the final hSC product was free of process-related contaminants [1]. Due to the autologous, per patient-derived nature of the investigational product, fresh organ donor tissue was used for the analysis of β1-heregulin and other process-related residuals such as mouse laminin, gentamicin, and bovine serum albumin [5, 13]. Briefly, connective tissue-free endoneurial fascicles from donor sural nerves were cultured for one week in β1-heregulin- and forskolin-supplemented medium. The predegenerated fibers were dissociated with collagenase and neutral protease to produce a cell suspension that was plated at low density in laminin-coated culture flasks for propagation. When the hSC cultures were ~70% confluent, the cells were enzymatically dissociated and re-plated for another round of subculture under identical conditions to generate pure, transplantation-grade hSCs. These cells were detached and rinsed three times using large volumes of serum-free DMEM/F12 medium before final resuspension in transplantation solution. Test samples for heregulin activity assays were collected from the hSC-conditioned medium (culture supernatant from the final hSC cultures) and from the washing steps identically during each process qualification run. The supernatants from rinses 2 and 3 were recovered for in-process testing prior to collection of the cell pellet (transplantable product). These samples were snap-frozen on dry ice and stored at − 80 °C until testing was performed.

The conditions tested in the assays were as follows: (1) DMEM/F12 with no additives (negative control); (2) freshly prepared hSC growth medium containing 10% FBS, 10 nM β1-heregulin peptide and 2 μM forskolin both provided from a stock solution (positive control); (3) growth medium diluted 10, 100, 1000, 10,000, and 100,000 times in DMEM/F12 (serial dilutions for estimation of dose-dependent changes); (4) Medium from semi-confluent hSC cultures before trypsinization (i.e., hSC conditioned medium), (5) 2nd and 3rd wash supernatants from hSC suspensions prepared as per transplantation protocols (test articles); and (6) 3rd wash supernatant spiked with 1:10 and 1:100 of fresh growth medium. Spiked samples were tested only in 2 out of 3 process qualification runs to control for potential heregulin-modifying elements in the final wash medium. All samples used to stimulate rat SCs were prepared in advance and subjected to one freeze-thaw cycle for consistency. Of note, the ErbB3-, ERK- and Akt-inducing activities were not notably different between frozen and fresh samples of the same culture medium (data not shown).

Plating, Starvation and Stimulation of Rat SCs for Heregulin Activity Assays

Cryovials of rat SCs were quickly thawed at 37 °C and re-suspended in 10–15 mL DMEM containing 10% FBS before collection by centrifugation. The cells were then plated directly onto PLL-coated 10-cm dishes at a density of 1–2 × 106 cells/dish in high-glucose DMEM, 10% FBS, 10 nM heregulin, 2 μM forskolin, and antibiotics [16]. Cells were cultured in a 37 °C humidified incubator set to 9% CO2 until they were 60–80% confluent (usually after 4–5 days) with regular media changes every 2–3 days. To obtain a single cell suspension, the cells were dislodged from the dishes via enzymatic treatment with trypsin/EDTA prepared in calcium- and magnesium-free HBSS. The cells were then plated into test dishes (24-well) coated with PLL and mouse laminin to promote strong adhesion of the rat SCs particularly during the starvation period. The rat SCs were seeded at a density of 100,000 cells per well in 0.5 mL DMEM containing 10% FBS (high serum) and immediately transferred to a CO2 incubator. Twenty-four hours later, the medium was replaced with an equivalent volume of HEPES-buffered DMEM containing 1% FBS (low serum), and the cells were  incubated for an additional 24-h period in a CO2 incubator. Sequential deprivation of mitogens and serum was required to prevent massive apoptotic cell death, as adult rat SCs are sensitive to abrupt changes in medium composition and pH, particularly after the removal of mitogens and serum [17]. Traces of serum and mitogenic factors were removed on the day of experimentation by replacing the medium with pre-warmed, serum-free DMEM/F12 (500 μl/well) followed by stabilization for ~ 1 h in a CO2 incubator. DMEM/F12 was chosen to both maintain the lowest possible endogenous kinase activity pre-stimulation and prevent pH changes. To initiate the assay, the medium was replaced expeditiously with 500 μl of the pre-warmed (37 °C) samples from the control and test articles, as described above, and the cells were transferred immediately to a CO2 incubator for the times indicated in the figures. To terminate the assays, the medium was removed by aspiration and the cells were lysed as described below. Each experimental condition was analyzed in duplicate samples with similar Western blot signals.

Further technical details on rat SC culturing and coating of 24-well dishes can be found in our previous publications [14, 16]. Briefly, a 0.02% PLL working solution was used to completely cover the surface of the wells for 1 h at room temperature (RT) before incubation for at least 1 h with a laminin solution prepared at a ratio of 0.4–0.6 μg laminin per cm2 of coated surface. The laminin solution was removed immediately before seeding the cells to ensure a fast adhesion and even distribution of the rat SCs as soon as 15–30 min post-plating. For consistency of results, we used passage 3–4 rat SCs from the same stock in all experiments.

Protein Lysate Preparation, Electrophoresis and Western Blot

Total cell lysates were prepared as previously described [12, 18, 19] with minor modifications. To expedite the cell lysis and maintain the phosphorylation signal as stable as possible at the time of sample collection, the culture medium was rapidly removed using a glass pipette connected to a vacuum line and the cells were lysed directly in the wells by the addition of 150 μl pre-warmed (50 °C) SDS-sample buffer (225 mM Tris/HCl, pH 6.8, 5% SDS, 50% glycerol, 250 mM dithiothreitol, 0.5 mg/ml bromophenol blue) for electrophoresis under denaturing conditions (SDS-PAGE). Protein lysates were transferred to 1.5 mL Eppendorf tubes expeditiously and denatured by boiling for 10 min in a heating block at 100 °C. Protein aliquots were resolved in denaturing (SDS) 10% polyacrylamide gels (1 mm thick, 15-lane gels) using15 μl of sample per lane. A colored molecular weight marker (Fermentas, Cat. # SM1811) was loaded in the first and last lanes of each gel as a reference for the consistent electrophoretic separation and transfer of the proteins. Electrophoresis was performed at a constant voltage of 80 V and stopped once the bromophenol blue dye reached the bottom of the gel. The average electrophoresis time under these conditions was 1.5–2 h. Fractionated proteins in the gels were transferred to polyvinylidene fluoride membranes (Immobilon-P membranes, Millipore, Bedford, MA Cat. # TM 151–1) using a standard liquid transfer protocol and a transfer time of ~ 2–3 h. Membranes were blocked for 30 min in Tris-buffer saline (TBS, Bio-Rad) containing 0.05% Tween-20 (TBS-T) and 2% ECL blocking agent (ECL Blocking Reagent, GE Healthcare Life Sciences, Cat. # RPN2125). The membranes were incubated with antibodies diluted in ECL blocking solution overnight (~ 20 h) at 4 °C, with continuous shaking. Next, the membranes were rinsed 3x with TBS-T and incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibodies (Goat anti-mouse IgG-HRP, Cat. # sc-2055, and goat anti-rabbit IgG-HRP, Cat. # sc-2301, Santa Cruz Biotechnology) in ECL blocking solution for 1 h at RT. The membranes were washed 3 times with TBS-T and immunoreactive proteins were detected by enhanced chemiluminescence (ECL) using ECL Plus detection reagents (GE Healthcare Life Sciences, Cat. # RPN2132) according to the manufacturer’s instructions. Light-sensitive film (Amersham Hyperfilm ECL, GE Healthcare Life Sciences, Cat. # 28–9068-39) was exposed according to the signal intensity of each blot, typically necessitating 1 to 30 min exposure time.

The electrophoresis and transfer equipment used was as follows: Mini PROTEAN II electrophoresis cell (Biorad, Cat # 165–2940), Mini Trans-Blot module (Bio-Rad, Cat. # 170–3935); and PowerPac Basic Power Supply (Bio-Rad, Cat. # 164–5050). The development of ECL membranes was performed using Automatic Film Processor, Konica Minolta, Model # SRX-101A. Gels were prepared in-house using 30% acrylamide / bis-acrylamide solution, 29:1, (Bio-Rad, Cat. # 1610156) and sodium dodecyl sulfate (SDS, Bio-Rad, Cat. #1610301). The electrophoresis, transfer and incubation buffers were prepared using the following reagents: 10x Tris/Glycine/SDS (Bio-Rad, Cat. # 161–0772); 10x Tris/Glycine buffer (Bio-Rad, Cat. # 161–0771); 10X TBS (Bio-Rad, Cat. # 1706435); Tween20 (Sigma, Cat. # P9416); Glycerol (Sigma, Cat. # G5516); 1,4 dithiothreitol (Roche, Cat. # 708984), bromophenol blue sodium salt (Sigma, Cat. # B8026). The antibodies used in heregulin activity assays were the following: pAkt-Ser-473 (Santa Cruz, Cat # sc7985, rabbit polyclonal, 1: 500); pERK1/2/MAPK (Santa Cruz, Cat # sc7383, mouse monoclonal, 1:500); pErbB3-Tyr-1289 (Cell Signaling, Cat # 4791S, rabbit monoclonal, 1:1000); Akt (Cell Signaling, Cat #. 9272, rabbit polyclonal, 1:1000); ERK2/MAPK (Santa Cruz, Cat # sc154, rabbit polyclonal, 1:500), and ErbB3 (Santa Cruz, cat # sc285, Rabbit polyclonal, 1:500). Other antibodies listed in the Materials section were used at 1:500.

Densitometric Analysis of Western Blot Bands

The relative optic density (O.D.) for each band was estimated using the gel analysis tool in ImageJ (NIH) available at (https://imagej.nih.gov/ij/). Scanned Western blot images (300 dpi) were transformed into grayscale mode and the background was digitally subtracted before analysis. The regions of interest were defined for each scan in sequential order, starting with the negative controls. The area under the curve method was used to quantify the intensity of the bands and to generate profile plots in which the height of the peaks represents the density of the bands in the original membranes. All quantified proteins were represented by discrete, single bands except for the 44/42 kDa doublet detected by P-ERK and total ERK antibodies. The 44/42 kDa doublets were enclosed within the same rectangular region of interest and quantified together for practical reasons. Data are represented as arbitrary O.D. units respective to the negative control value, or as the ratio between the phosphorylated and total protein bands for each protein species to obtain the relative level of phosphorylation at each condition. Images with uneven or interrupted bands were not used for densitometric quantification.

Cell Proliferation Assays

The incorporation of the thymidine analog BrdU into nuclear DNA was assayed as a measure of S-phase entry, as previously described [12]. Briefly, adult rat SCs were plated on PLL/laminin-coated 24-well dishes (50,000–70,000 cells/well) in DMEM supplemented with 10% FBS. One day later, the medium was changed to HEPES-buffered DMEM containing 1% FBS (non-proliferating medium), to induce quiescence. The cells were stimulated with mitogenic factors in the absence or presence of 10 μM of 4557 W (referred to as ErbB2 inhibitor or ErbB2i) for 3 days in medium containing BrdU (1 μM). Quiescent rat SCs typically incorporate BrdU within 24–48 hs after heregulin stimulation. To detect incorporated BrdU, the cells were fixed sequentially with 4% paraformaldehyde and − 20 °C methanol, and blocked with 5% normal goat serum. Cells were treated for 30 min with a 50% solution of DNase in the presence of anti-BrdU (1:300) and then incubated with Alexa594-conjugated secondary antibodies. BrdU staining was performed alone or together with SC markers.

Immunofluorescence Microscopy

The cells were double fixed with paraformaldehyde and methanol, as described above, and blocked with 5% normal goat serum in PBS before the addition of antibodies against P-ERK (1:200) or GFAP (1:300) and incubation overnight at 4 °C. The cells were washed with PBS, incubated with fluorescent secondary antibodies (1:300) prepared in 5% normal goat serum containing the nuclear stain DAPI, and mounted for microscopy using Vectashield. Images were taken using a cooled digital CCD camera (SensiCam QE, Cooke Corp.) coupled to an Olympus IX70 inverted fluorescence microscope. Black and white images (8-bit, tiff format) were artificially colorized in RGB format, digitally processed and arranged for presentation using Adobe Photoshop 21.0.2 and Adobe Illustrator 24.0.1.

Statistical Analysis

Statistical comparisons were performed using GraphPad Prism software, Version 4 (GraphPad Software, Inc., San Diego, CA, USA). Experimental data are expressed as the mean ± standard error of the mean (SEM) of samples from 3 independent experiments. One–way ANOVA was used followed by Bonferroni’s post hoc test. The results were considered significant when * p < 0.05.

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