A novel human leiomyoma tissue derived matrix for cell culture studies

Human uterus leiomyoma tissue (after taking samples for histopathological analyses) was received from the Oulu University Hospital, Department of Gynecology [1] and the Tampere University Hospital, Department of Gynecology after obtaining the patients’ written informed consent. The use of myoma tissue was approved by the Ethics Committee of both the Oulu University Hospital, and the Tampere University Hospital. Myogel was prepared according to the method described [9] for the preparation of EHS sarcoma derived Matrigel® (BD Biosciences), with minor modifications. Briefly, myoma tissue, frozen with liquid nitrogen, was ground to a powder with a CryoMill (Retsch, Haan, Germany), and 10 g of tissue powder was suspended in 20 ml of ice cold 3.4 M, pH 7.4 NaCl buffer. After centrifugation, the pellet was homogenized in another 20 ml of the same NaCl buffer using a T18 Ultra-Turrax (IKA®-Werke GmbH & Co. KG, Staufen, Germany). A T18 Ultra-Turrax was also used in all the following homogenizations. The protein concentration in each preparation was measured using a DC Protein Assay (Bio-Rad) according to the manufacturer’s instructions. The absorbance at 590 nm was measured using a Victor³V 1420 Multilabel Counter and Wallac 1420 Manager (Perkin Elmer Life and Analytical Sciences, Turku, Finland). The protein concentrations in various Myogel batches were diluted using cell culture media described in the cell culture -section (see below) to match those of Matrigel®. The Myogel solution was then stored in small (≤1 ml) aliquots at −20 °C.

Samples (20 μg) of four different Myogel batches (preparation numbers 12, 15, 16 and 17) and their mixture, as well as four different Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234) samples were loaded on a gradient (4 %, 8 %, 15 %) SDS-PAGE gel and separated using 15 mA for 90 min. A PageRuler Plus Prestained Protein Ladder (Thermo Scientific) was used as a molecular weight marker. Proteins were stained using Coomassie Blue and the gel was washed with elution buffer to remove excess staining. The gel was photographed over a stripping table.

For proteomic analysis, 10 μg and 20 μg of four different Myogel batches (preparation numbers 3, 4, 6, 9) were separated as above. The gel was viewed over a stripping table, and individual bands were cut and collected from the gel. After digestion (0.3 μg of trypsin was used/band) each band was resuspended with formic acid (see μl/band in the Additional file 1: Figure S1B) on three selected Myogel samples (preparations 3, 6, 9) and stored in −20 °C until gel digestion for mass spectrometry. The gel digestion of selected samples for mass spectrometry was done according to Hanna et al. [25] with some modifications.

For the proteomic analyses two Myogel and one Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234) samples were further purified by buffer exchange using an Amicon Ultra ultrafiltration unit with a 10 kDa cutoff (Millipore) and urea buffer (7 M urea, 2 M thiourea, 4 % [w/v] CHAPS, 30 mM Tris, pH 8.5). After that the protein samples were sonicated and centrifuged. The protein concentrations in the supernatants were determined in duplicate with a Bradford-based assay according to the manufacturer’s instructions (Roti®-Nanoquant, Carl Roth) with urea buffer as a control and aliquots were stored at −20 °C. For 2-DE, 100 μg of the protein solution of each sample was adjusted with rehydration urea buffer (7 M urea, 2 M thiourea, 4 % [w/v] CHAPS, 0.15 % [w/v] DTT, 0.5 % [v/v] carrier ampholytes 3–10, Complete Mini protease inhibitor cocktail (Roche)) to a final volume of 400 μl. In-gel rehydration with IPG strips (pH 4–7, 18 cm, GE Healthcare) was performed overnight. Isoelectric focusing (IEF) was carried out with the Multiphor II system (GE Healthcare) under paraffin oil for 55 kVh. SDS-PAGE was performed overnight in polyacrylamide gels (12.5 % T, 2.6 % C) with the Ettan DALT II system (GE Healthcare) at 1–2 W per gel and 12 °C. The gels were silver stained as described by Ohlmeier et al. [26] and analyzed with the 2-D PAGE image analysis software Melanie 3.0 (GeneBio).

Gelatinolytic enzymes in Myogel and Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234) were detected by a zymography method using fluorescently labeled gelatin [27]. Prestained low-range SDS-PAGE Standards (Bio-Rad) as well as purified control MMP-2 and MMP −9 samples were loaded in adjacent wells. After electrophoresis, gelatinases were activated by incubating the gels with zymography buffer (50 mM Tris–HCl, 5 mM CaCl₂, 1 μM ZnCl₂, 0.02 % NaN³, pH 7.5) overnight at 37 °C. Gelatin degradation was visualized under long wave UV light and photographed using an AlphaDigiDoc® RT Gel Documentation System (Alpha Innotech, San Leandro, CA).

For pH comparison, Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234) was diluted with an equal volume of serum free medium, and the same amount of total protein for Myogel was obtained by diluting it 10 + 6 with serum free medium. The pH of both gels was measured at the beginning, after 17 h, and at the end of the 48 h experiment. The gels were incubated at 37 °C in a 5 % CO₂ humidified cell culture chamber with or without HSC-3 cells (see below for the culture conditions) on top of the gels.

Aggressive human oral tongue squamous cell carcinoma cell line HSC-3 (Japan Health Sciences Foundation, Japan) was cultured in a 1:1 DMEM/F-12 medium (Life Technologies) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml fungizone, 50 μg/ml ascorbic acid and 0.4 μg/ml hydrocortisone (all from Sigma-Aldrich) and 10 % heat inactivated fetal bovine serum (FBS; Life technologies). HSC-3 cells labeled with GFP were generated by stable transduction with non-silencing GIPZ lentiviral shRNAmir control particles (pGIPZ vector contains GFP in order to track shRNAmir expression; Thermo Fischer Open Biosystems) with puromycin (Sigma-Aldrich) selection according to the manufacturer’s instructions. Nuclear histone-2B (H2B)-coupled mCherry expression vector pLenti6.2 V5/DEST (a gift from Dr. Cindy E. Dieteren, Department of Cell Biology, Radboud UMC, Netherlands) was introduced into the HSC-3 cells with cytosolic GFP labeling using lentivirus mediated infection and selected in culture media containing 5 μg/ml blasticidin-S (Merck Millipore). HSC-3 cells expressing cytoplasmic GFP and H2B-coupled mCherry were cultured in DMEM/F12 medium (Gibco/Thermo Fisher Scientific), 10 % heat-inactivated FBS (HyClone/Thermo Fisher Scientific), 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich), 50 μg/ml L-ascorbic acid (Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich). HSC-3 cells labeled with RFP were generated by stable transduction with commercial lentiviral particles containing a non-coding control sequence (Amsbio) and selected with puromycin. They were cultured as normal HSC-3 cells.

The SCC-9 cell line (American Type Culture Collection, ATCC) was maintained in DMEM/F-12 medium (Invitrogen) supplemented with 10 % FBS (Cultilab), 400 ng/mL hydrocortisone, and antibiotic/antimycotic solution (Invitrogen). SCC-9 cells were labeled with ZsGreen protein and implanted subcutaneously into the footpads of the left front limb of BALB/c nude mice and LN-1 and LN-2 cell lines with increased metastatic potential were derived by in vivo selection from axillary lymph nodes with metastatic cells as described earlier [6]. Both LN-1 and LN-2 cells were maintained in culture as SCC-9.

Normal oral gingival fibroblasts (GF) were established from palatal gingiva mucosa biopsies and cultured in DMEM medium (high glucose, GlutaMAX^TM and pyruvate) supplemented with 10 % FBS, 50 U/ml penicillin, 50 μg/ml streptomycin and 2.5 μg/ml amphotericin B (all from Gibco). After obtaining written informed consent, the palatal tissue biopsies were taken from healthy volunteers for another study to be used as a starting material for control fibroblast cell line cultures. The volunteer consent encompassed the use of obtained cell lines for other studies as well. The use of palatal tissue was approved by the Ethics Committee of the Helsinki University Hospital. The carcinoma associated fibroblast (CAF) cell lines were generated from fragments of tongue squamous cell carcinomas by using tissue explants [28]. They were cultured in DMEM medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml ascorbic acid, 250 ng/ml fungizone, 1 mmol/L sodium pyruvate (Sigma-Aldrich) and 10 % heat inactivated FBS.

Melanoma cell lines SK-Mel-25 and A2058 (ATCC) were maintained in RPMI medium (Invitrogen) supplemented with 10 % FBS (Cultilab) as described earlier [29]. Human umbilical vein endothelial cells (HUVEC, ATCC) were cultured in a 1:1 mixture of DMEM/F12 medium (Invitrogen) supplemented with 10 % FBS and 400 ng/ml hydrocortisone (Sigma-Aldrich).

All the cells were cultured in a humidified atmosphere of 5 % CO₂ at 37 °C and passaged routinely using trypsin-EDTA (Sigma-Aldrich). The media were changed every 2–3 days. They were regularly tested and confirmed to be negative for mycoplasma infection using a MycoTrace PCR Detection Kit (PAA Laboratories GmbH). Cell line identity was not routinely performed.

A cell adhesion assay was conducted to determine how many cells bind to Myogel compared to Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234). In this assay, HSC-3 cells were cultured to subconfluence. Wells in a 96-well plate were coated for 24 h either with 100 μl of PBS, BSA (bovine serum albumin, 10 μg/ml, Sigma-Aldrich), Matrigel® or Myogel (two different batches). Matrigel® was diluted to 1:10 in PBS and Myogel was diluted to the same protein concentration. At the same time, the cell culture medium was changed to serum-free medium. The next day the excess liquids were removed and the culture plates were incubated with 100 μl/well of 0.1 % BSA for 2 h and washed with PBS. HSC-3 cells (6000) in 100 μl of serum-free medium were added to each well and the wells were incubated at 37 °C in a 5 % CO₂ humidified atmosphere for 2 h. The non-adherent cells were rinsed off, and the remaining cells were fixed with 10 % trichloroacetic acid (TCA), stained with crystal violet and quantified using an ELISA reader at 540 nm.

Adhesion of GFs on top of Myogel was studied using 6-well plates coated at 37 °C in a 5 % CO₂ humidified atmosphere with 0.62 mg/ml Myogel diluted with DMEM without supplements. After 2 h the excess liquids were removed and 150,000 GFs were added in their normal culture medium. The cultures were photographed with Olympus CKX41 inverted microscope (Hamburg, Germany) after 2.5 h and 9.5 h with 20 x magnification to record the morphology of the cells.

An 8-well Nunc™ Lab-Tek™ chambered slide (Thermo Scientific) was used for the experiment. Wells were coated with either rat tail collagen type I (BD Biosciences), one of three different batches of Myogel or an equal (50–50 %) mixture of collagen type I and one type of Myogel. A total of 150 μl of coating mixtures were prepared, with a final protein concentration of each gel mixture of 0.62 mg/ml, which were adjusted with the addition of culture medium. Two wells were not coated and were kept for positive and negative controls for the subsequent TUNEL assay. The Lab-Tek™ chamber slide was covered and placed in the incubator at 37 °C in 5 % CO₂ for 2 h. Subsequently, 5000 CAFs were added on each well and the slide placed in the incubator overnight. On the following day, the wells were washed twice with PBS, air-dried for few min and fixed with freshly prepared 4 % (w/v) paraformaldehyde in PBS (pH 7.4) for 1 hour at RT. After 2 washes with PBS for 5 min each, the TUNEL assay was performed using a commercially available kit (In Situ Cell Death Detection Kit, Roche). At the end of the assay, samples were counterstained with DAPI for 10 min at RT. After coverslip mounting with a water-based medium, 100 cells were counted under a confocal microscope using the blue channel (405 nm) and apoptosis was detected using the green channel (488 nm).

Myogel for CAF embedding was prepared as described for collagen organotypic culture in Nurmenniemi et al. [1]. Briefly, a gel mixture including 8 volumes of Myogel (4.5 mg/ml), 1 volume of 10 × DMEM (Sigma Aldrich) and 1 volume of FBS with CAFs (final concentration 70,000 cells/ 400 μl gel mixture) was prepared on ice and 400 μl aliquots were added into the wells of a 48-well plate. After 40 min polymerization, 100 μl of complete CAF medium was added on the polymerized gels. The plates were incubated at 37 °C in a 5 % CO₂ humidified atmosphere. Cells were fed twice a week and pictures were taken with an EVOS microscope (Advanced Microscopy Group, Bothell, WA) weekly for three weeks.

For the soft agar mimicking assay, one percent sterile base low melting agarose (LMA, Sea Plaque Low Melting Agarose, Lonza) melted in PBS was mixed with 10x DMEM/F12, 100 % FBS to give a final 0.8 % agarose with 1x medium, 10 % FBS. One-half ml of the mixture was added to wells in a 24-well plate and allowed to solidify for at least 30 min in the laminar flow hood. 10 000 HSC-3 (H2B-GFP) cells in 50 μl of FBS were mixed with 50 μl of 10x DMEM/F12 and 0.4 ml of 0.5 % LMA (as a control, final agarose concentration 0.4 %) or with 50 μl of 10x DMEM/F12, 0.2 ml of 1.0 % agarose and 0.2 ml Myogel (final agarose concentration 0.4 %, final Myogel protein concentration 2.2 mg/ml). Myogel was centrifuged at 4000 rpm for 10 min prior to the procedure. The agarose mixture was gently mixed by swirling, and 0.5 ml was added on the top of the agarose base. The plates were incubated at 37 °C in a 5 % CO₂ humidified atmosphere for 28 days. The cells were fed twice a week with 0.25 ml normal HSC-3 medium. Pictures of the colonies were taken with transmitted light and in the GFP & RFP channels in different objectives (10x, 20x & 40x) using an EVOS inverted microscope. Cells in colonies were calculated from the pictures and ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA) was used to measure colony area.

The spheroids were formed according to published protocol [30]. 20 μl drops of the cell suspension (70,000 HSC-3 (H2B-GFP) cells per drop) suspended in DMEM/F12 medium with 10 % FBS were placed onto the lids of 10 cm dishes, which were inverted over dishes containing 10 ml PBS. Hanging drop cultures were incubated for 72 h, the resulting cellular aggregates were harvested by a pipette and embedded in two different conditions (Myogel-LMA & LMA) into the wells of a 48- well plate as in the soft agar colony formation –assay above. After 6 h incubation at 37 °C in a 5 % CO₂ humidified atmosphere, 150 μl normal HSC-3 cell culture medium was added per well. Pictures of the spheroids were taken with 4x objective using an EVOS inverted microscope at 0 h, 24 h, 48 h and 72 h after embedding. ImageJ software was used to measure spheroid area and the results were calculated as a ratio to the area of the implanted spheroid right after embedding (without media).

For microarray analysis, 90,000 HSC-3 cells transduced with RFP were seeded into uncoated or Myogel coated 6-well plates (three wells each). The next day the cells were harvested for RNA extraction using a Qiagen RNA kit. Three samples of each group (on top of plastic or Myogel coating) were pooled; the pools contained an equal amount of RNA from each sample. Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays were used for microarray analysis and experimental procedures were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 1 μg of total RNA was used as a template to synthesize biotinylated cRNA by means of the GeneChip 3’IVT Express kit (Affymetrix) according to the manufacturer’s instructions. The cRNA was fragmented to 35 to 200 nt prior to hybridization to Affymetrix Human Genome U133 Plus 2.0 arrays containing approximately 55,000 human transcripts. The array was washed and stained with streptavidin-phycoerythrin (Molecular Probes). Finally, biotinylated anti-streptavidin (Vector Laboratories Inc.) was used to amplify the staining signal and a second staining was performed with streptavidin-phycoerythrin. The arrays were scanned on a GeneChip Scanner 3000 (Affymetrix, High Wycombe, United Kingdom). The expression data was analyzed to find genes with fold changes (FC) of 1.5 or more using dChip software [31]. The genes with FC 1.2 or more were divided into Gene Ontology (GO) categories using a dChip enrichment analysis tool.

To analyze the effects of Myogel and Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234) on the migration of HSC-3 cells, 24-well plates were coated with 0.62 mg/ml Myogel (two different batches) or 0.62 mg/ml Matrigel®. The coating was left to solidify for 2 h at 37 °C and then washed twice with PBS. HSC-3 cells (90,000) were allowed to attach overnight, and were then wounded with a pipette tip, rinsed twice with PBS, post-coated for 1 h and rinsed before 1 % FBS medium was added. The wounds were photographed with an EVOS photomicroscope at 0 h and 24 h after scratching. The area of the open wound was measured using ImageJ software and the results were calculated as a percentage of wound closure.

To compare Myogel with Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234) as an invasion assay material, experiments were carried out according to BD Biosciences instructions to coat the filter membranes with Matrigel®. Fifty μl of either 1 + 1 Matrigel® or Myogel (three different batches at the same protein concentration as Matrigel®) diluted with serum-free medium was added to the upper chamber of a Transwell® nylon filter membrane insert (Corning Inc.), incubated at 37 °C in a 5 % CO₂ humidified atmosphere for 30 min after which 50,000 HSC-3 cells suspended in 100 μl of serum-free medium were seeded onto the upper compartment of the Transwell® chambers. The Transwell® inserts were incubated for 12 h - 48 h at 37 °C in a 5 % CO₂ humidified atmosphere, after which the cells were fixed in 10 % TCA for 15 min, rinsed and air-dried overnight. Once dry the membranes were stained with crystal violet for 20 min and the excess stain was removed by water rinsing. The non-invasive cells from the upper side of the membrane were removed by carefully sweeping with a cotton swab. Next, the membranes were removed from the inserts and placed on microscope slides and the number of invaded cells through Myogel and Matrigel® were counted. We also tested different mixtures of Myogel and Matrigel® (2 + 1, 1 + 1 and 1 + 2) in the invasion assay.

We used a final concentration of 0.2 % LMA in 3.2 mg/ml final Myogel protein concentration to solidify Myogel for invasion assays. To compare the results with Matrigel® we diluted Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234) 1 + 4 (final protein concentration 2.0 mg/ml) with the same concentration of 0.2 % agarose. Serum-free cell culture medium was used for gel dilution. Fifty μl of the Myogel-LMA or Matrigel agarose mixtures was added on the upper chamber of Transwell® nylon filter membrane insert, incubated ½ h at RT and thereafter at 37 °C in a 5 % CO₂ humidified atmosphere until the cells were ready to be seeded on the top of the gel. HSC-3 cells were trypsinized and counted, trypsin inhibitor instead of serum-containing medium was used to inactivate trypsin. Five hundred μl of 10 % serum containing medium was added into the lower chamber of the Transwell®, and 50,000 HSC-3 cells suspended into 100 μl of medium containing 0.5 % lactalbumin instead of serum were seeded into the upper compartment of the Transwell® chamber. The cells were allowed to invade for one to three days and the invasion was quantified by staining the cells with crystal violet followed by counting the cell number. In this preliminary assay we tested different agaroses and concentrations with only one Transwell® in each condition and 0.2 % LMA was chosen for further experiments. In another set of experiments, the invasion of SCC-9, LN-1, LN-2, HSC-3, SK-Mel-25 and A2058 cells was studied in Myogel-LMA and growth factor-reduced Matrigel® (Matrigel®-GFR, BD Matrigel Matrix, BD Biosciences, Cat. Number 35430) diluted 1 + 1 with serum free medium without LMA. The invasion was quantified with Toluidine Blue -staining. Briefly, after 72 h invasion, the cells were fixed with 4 % formaldehyde for 1 h at RT, washed once with PBS, stained for 5–10 min in Toluidine blue solution (filtered 1 % Toluidine Blue + 1 % disodium tetraborate in ddH₂0) at RT, excess dye was rinsed out with deionized water, excess gel and the cells from the upper side of the membrane were removed by using cotton swabs and when necessary, excess dye was further removed from the inside and outside of the Transwell® inserts with cotton swabs immersed in a solution of 1:1 water/ethanol. The dye was eluted by dipping the Transwell® inserts in a solution of 1 % SDS (500 μl). The 1 % SDS solution containing the eluted dye was transferred into a 96-well plate and the absorbance was measured at 650 nm.

The gel from rat tail type I collagen (BD Biosciences) was prepared according to the manufacturer’s protocol, with a final collagen concentration of 1.7 mg/ml. The collagen/Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234) (1.5 mg/ml, 1.5 mg/ml) and collagen/Myogel (1.5 mg/ml, 4.3 mg/ml) mixtures were prepared similarly. The hanging drop technique was used to observe the cell movement under 3D culture conditions. HSC-3 (H2B-GFP) cells were washed with PBS, trypsinized and 70,000 cells in 10 μl of DMEM/F12 medium (2 % FBS) were mixed with 50 μl of the matrix mixture. Twenty μl of the cell suspension in each matrix was dropped on the 4 compartment plate. The plate was inverted after 5 min incubation in culturing conditions and incubated for 3 h in a humidified chamber in culturing conditions. Mimosine (200 μM) was added to the medium to synchronize the cell cycle. Images were taken with a Zeiss Axio Observer.Z1 with a EC Plan-Neofluar 40x/0.75 M27 objective (Göttingen, Germany). Images consisting of 1024 x 1024 pixels were taken every 15 min with a 19.8 μm Z-stack volume (0.6 μm thickness) with a Hamamatsu Camera#2 controlled by Zeiss Zen Blue software (Zeiss) for 20 h. This assay was performed twice. The analysis of the cells in hanging drops is described in the Additional file 2: Supplementary Method.

SPSS for Windows software version 21.0 (IBM) or GraphPad Prism 6 (GraphPad Software) were used for statistical analyses. To establish the statistical significance of differences between the two independent cell culture groups, a Mann–Whitney U test was used to compare the groups.

The reproducibility of different Myogel batches was first investigated in Coomassie Blue stained gradient SDS-PAGE gels. Different Myogel preparations and the mixture of various batches showed relatively similar protein band patterns, and only slight variations in the intensities of differently sized bands were visible (Additional file 1: Figures S1A and S1B). Two Myogel batches were further investigated by 2-DE (Additional file 1: Figure S1C) which confirmed with almost similar protein patterns in the silver stained 2D gels likewise the high reproducibility of these Myogel preparations.

After that the protein compositions of Myogel and Matrigel® were compared. In SDS-PAGE gels more bands were detected for the Myogel (Additional file 1: Figure S1A) and also the 2-DE separation showed a significant difference between Matrigel® and Myogel samples (Additional file 1: Figure S1C). If similar protein amounts (100 μg) were separated significantly more spots were visible for the Myogel. Increasing the amount of Matrigel® proteins to 300 μg resulted in a significantly higher number of detectable spots (Additional file 1: Figure S1C). This suggests that a few high abundant proteins, including these visible in the gel with lower protein amount, comprise a major part of the Matrigel® proteome. However, also with higher protein amounts Matrigel® and Myogel showed still distinct protein patterns. The here shown differences between Myogel and Matrigel® proteomes are possibly due to the difference between species (human vs. mouse) and the nature of the starting material (leiomyoma vs. sarcoma).

We next analyzed the protein content of Myogel and compared it with the published data of Matrigel® [32‐34]. For further proteomic analyses, different Myogel batches were separated by gradient SDS-PAGE (Additional file 1: Figure S1B). Since Myogel batch number 4 differed most from the other three batches (3, 6 and 9; Additional file 1: Figure S1B), it was omitted from the mass spectrometry analysis. Two of the Myogel samples gave successful results, and altogether 765 proteins were identified (Additional file 3: Table S1). Among the Myogel proteins, 34 % (259 proteins) were the same as in Matrigel® where 1030 proteins were identified. Based on the comparison, for instance laminin, type IV collagen, heparan sulfate proteoglycans, nidogen and epidermal growth factor were found in both. Based on mass spectrometry analysis, Myogel lacked enactin, which is present in Matrigel®, but contained for example tenascin-C, collagen types XII and XIV, etc., which were lacking in Matrigel®. Based on zymography, Myogel contained both latent and active forms of MMP-2, whereas in Matrigel® latent and active forms of both MMP-2 and MMP-9 were present (Additional file 1: Figure S1D) as shown earlier [35].

The pH of Myogel was initially between 7.0 and 7.5, and after 48 h incubation the pH remained the same. The pH of Myogel with HSC-3 cells on top dropped slightly from 7.0–7.5 to 6.5–7.0 (0 h and 48 h incubations, respectively), whereas in Matrigel® the pH at the same time dropped from a slightly alkali 8.0–8.5 to as low as 6.0–6.5 (Additional file 4: Table S2). This indicates that Myogel samples are closer than Matrigel® to neutral pH, and Myogel keeps the pH more stable than Matrigel® during cancer cells culture experiments.

HSC-3 cells adhered significantly more to plates pre-coated with Myogel than with BSA, or to the plain plates kept in PBS. However, they adhered even more readily to Matrigel® coated plates (Fig. 1). The adhesion experiment with gingival fibroblasts (GFs) showed that after 9.5 h incubation on top of Myogel, fibroblasts were well spread and vital (not shown). Based on the TUNEL assay, from 98 to 99 % of the carcinoma associated fibroblasts (CAFs) seeded on top of various batches of Myogel were alive even after 24 h incubation (not shown). However, when CAFs were embedded within the Myogel matrix, almost all died within 21 days (not shown). In contrast, embedded HSC-3 cells stayed alive up to 28 days, divided and formed colonies within Myogel combined with LMA. The results with HSC-3 cells were relatively similar using either Myogel-LMA or a conventional soft agarose (LMA) assay (Figs. 2a and b); in Myogel-LMA the colony number was six percent less than in LMA (47 vs. 50). However, more colonies with the lowest cell number were present in LMA, while the highest cell number/colony was present in Myogel-LMA (Fig. 2b). According to nuclear RFP expression, 92 % of the cancer cells were alive in Myogel-LMA colonies, whereas 85 % were alive in LMA colonies. The total average area of cell colonies in Myogel-LMA was three percent less than in LMA (not shown). In HSC-3 cell hanging drop spheroid cultures the area of the spheroids embedded in Myogel-LMA grew more in 24 h incubation than in spheroids embedded in plain LMA (Fig. 2c and d). The area growing rate remained higher in Myogel-LMA even during 72 h follow-up and the spheroid area enlarged more in ratio to 0 h area in Myogel-LMA cultures than in plain LMA cultures (Fig. 2c and d). On average the increase at 72 h in ratio to 0 h was 21.5 % in Myogel-LMA and 12.2 % in plain LMA. In the most enlarged spheroids, the area increase at 72 h in Myoogel-LMA was 63.5 % while in plain LMA it was only 43.1 % (Fig. 2d). In less enlarged spheroids, the average increase at 72 h in Myogel-LMA was 11.0 % whereas in plain LMA it was 4.5 % compared to 0 h spheroids (Fig. 2d).

Gene expression assays are usually done with cells grown on tissue culture plastic. We wanted to see if Myogel coating has an effect on expressed genes in HSC-3 cells compared to cells grown on plain plastic. Approximately 1.4 % of the genes (totally 751) were differentially expressed (FC 1.5 or more) when the HSC-3 cells cultured on the top of plastic were compared to the corresponding cells grown on Myogel (raw data presented in Additional file 5: Table S3 showing 807 probes). When the genes were divided into groups according to their biological function we found that the Myogel coating affected those genes that are related to intracellular organelle and cytoskeleton organization and biogenesis. A significant change in pathway analysis was found only in the G13 signaling pathway that is related to actin polymerization and reorganization (www.​genmapp.​org).

In the scratch assay, HSC-3 cells migrated significantly more in Myogel coated wells than in wells coated with Matrigel® (Fig. 3a and b). However, in uncoated wells their migration was faster than in coated wells (Fig. 3a and b).

In order to test the use of Myogel on cancer invasion Transwell® -assays, we first compared the invasion of the most aggressive oral tongue carcinoma cell line (HCS-3) using Myogel and Matrigel®. The HSC-3 cells invaded significantly more efficiently through Myogel than Matrigel® (Fig. 4a). Invasion varied slightly in different Myogel batches, but HSC-3 cells invaded in all Myogel samples more than in Matrigel® (Fig. 4b). When Myogel and Matrigel® were mixed, HSC-3 cells invaded more efficiently when the mixture contained more Myogel, and less when the portion of Matrigel® increased (Fig. 4c). The invasion pattern of HSC-3 cells was different in Myogel and Matrigel®. The cells invaded more evenly throughout the whole Transwell® membrane in Myogel coated filters, whereas in Matrigel® experiments they invaded more in clumps (Fig. 4d).

Since Myogel batches vary slightly in their protein content, as seen in the gradient gel in Additional file 1: Figures S1A and S1B, we created a more homogenous, reliable and easier to handle matrix by adding low-melting agarose into the Myogel mixture. We noticed that HSC-3 cells did not invade through a plain agarose coated membrane, but they invaded through a Myogel-agarose (Myogel-LMA) mixture (Fig. 5a and b). Interestingly, the passage number of the HSC-3 cells seemed to affect the invasion through Myogel- and Matrigel®-mixtures; cells with higher passages invaded slightly less through Matrigel® mixtures than through Myogel-containing mixtures, whereas cells with a low passage number invaded slightly less through Myogel-LMA than through Matrigel®-LMA (Fig. 5a). The invasion pattern of HSC-3 cells through agarose-containing mixtures was similar to that observed in plain Myogel or Matrigel®; in Myogel-LMA mixtures cells invaded more evenly throughout the whole Transwell® membrane than in Matrigel®-LMA mixtures (Fig. 5b).

In another experiment, Myogel-LMA was compared with Matrigel®-GFR. In general, all the oral squamous cell carcinoma cell lines used for these invasion assays (HSC-3, LN-1, LN-2 and SCC-9) preferred Myogel-LMA to Matrigel®-GFR during the invasion (Fig. 5c). HSC-3 and LN-2 (in this order) seemed to have a higher potential to invade in both Myogel-LMA and Matrigel®-GFR, compared to the LN-1 and SSC-9 cell lines. The cells invading the Myogel-LMA seemed to keep more of the morphological characteristics observed in monolayer cultures. In contrast, the cells grouped together as they invaded the Matrigel®-GFR matrix, and lost the “fusiform-starshape” characteristic of oral carcinoma cell lines (not shown). In addition to oral carcinoma cell lines, we tested Myogel-LMA on SK-Mel and A2058 melanoma cell lines and found that also they invaded Myogel-LMA more efficiently than Matrigel®-GFR (Fig. 5c).

To observe the cell behavior in 3D culture in different matrices, we have established an optimized hanging drop method modified from the previously reported protocol [36]. HSC-3 cells moved in a relatively similar fashion in pure collagen, Myogel-collagen and Matrigel®-collagen matrices (Additional file 6: Movie S1, Additional file 7: Movie S2 and Additional file 8: Movie S3). However, interestingly the speed of the cells was highest in the Myogel-collagen matrix and lowest in the Matrigel®-collagen matrix (Additional file 1: Figure S2, Additional file 6: Movie S1, Additional file 7: Movie S2 and Additional file 8: Movie S3).

In vitro angiogenesis assays are commonly used to assess pro- or anti-angiogenic drug properties [37]. Here, tube formation could be quantified in all matrix-assays tested (Fig. 6a). With Myogel-LMA, tube formation was visible after 12 h, and even after 72 h the endothelial cells were alive (Fig. 6a), unlike in Matrigel®-GFR or ECMatrix™ (not shown), where most of the HUVEC cells were already apoptotic after 24 h. We found that the number of tubules formed was three times higher in Myogel-LMA compared to ECMatrix™ (Fig. 6b). Otherwise, measuring the diameters of the capillaries, the tubule parameters in Matrigel®-GFR, and especially in ECMatrix™ assays, were significantly higher than in tubes formed in Myogel-LMA (Fig. 6c).