Keywords

1 Introduction

Basement membranes (BMs) are specialized extracellular sheet-like matrices underlying epithelia in all mammals [1]. They are key elements during embryogenesis and are mainly composed of laminins, collagen-4 and heparin sulfate proteoglycans [2], joined together by nidogens, perlecans and other proteins [3].

In this regard, the primary function of laminins, a family of large heterotrimeric (a, ß, γ) glycoproteins present in BMs, is to interact with receptors anchored in the plasma membrane of cells, such as endothelial or neuronal cells [1]. Laminin-111, a 800-kDa protein, is the prototype of the family and the best characterized laminin isoform [1, 3] It is adhesive for most cell types, promotes cell survival in vitro and has various biological key activities [3,4,5], including cell adhesion, proliferation, differentiation and migration [1, 6]. Laminins are frequently used for in vitro and in vivo neuronal cell cultivation [7,8,9,10,11], angiogenesis [5, 12], wound healing [6, 13,14,15], or stem cell studies [16, 17].

Laminin-111 was the first laminin type isolated by Ruppert Timpl from Engelbreth-Holmes Sarcoma (EHS) mouse material during the 1970s [18]. For several years this has been the only known laminin isoform [19]. Since its discovery, many attempts have been made to isolate laminin-111 from a human source such as placenta [20,21,22,23,24] or produce it recombinantly [25, 26]. However, no human equivalent to the mouse tumor derived EHS laminin-111 is available for large-scale production and therefore, more than 30 years after its discovery, laminin-111 extracted from xenogenic EHS tumor tissue is still the frequently used gold standard for various in vitro and in vivo research protocols [27].

The aim of this study was to establish an effective method for isolation of human placental laminin-111 (pLm-111). The method was based on an extraction step via Tris-NaCl buffer to yield a laminin-rich protein fraction, followed by a protein precipitation step using 30% ammonium chloride combined with a series of diafiltration and salt precipitation steps to remove non-laminin contaminants and therefore purify the laminin-111 isolates. The resulting purified laminin-111 was biochemically characterized using a combination of dot blot, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and HPLC-based amino acid analysis. The in vitro biocompatibility and bioactivity of laminin-111 was demonstrated using NG 108-15 neuronal cell lines, Schwann cells and GFP-expressing human umbilical vein endothelial cells (gfpHUVEC).

2 Materials and Methods

If not stated otherwise all chemicals were purchased from Sigma Aldrich and of analytical grade.

2.1 Collection of Human Placenta Tissue

Placenta material was collected after caesarian section from the Landes-Kinderklinik Hospital Linz, Austria (with the permission of the local ethical board and informed consent from all donors), delivered to LBI Trauma laboratories on dry ice, and stored at −20 °C until the isolation procedure was performed.

2.2 Isolation Procedure of Placenta Laminin-111 (pLm-111)

All isolation steps were performed in a cold-room at 4 °C. For all diafiltration steps in this protocol the tangential flow filtration (TFF) Ultralab™ system PALL (VWR, Vienna, Austria) has been used, equipped with a 100 kDa cut-off Ultrasette™ tangential flow filter.

After thawing, the placenta was dissected free of the outer membranes, amnion and chorion as well as of the umbilical cord. The residual basal tissue was used for the isolation process. Blood components were removed by repetitive homogenization steps of 100 g basal placenta tissue in 200 mL phosphate buffered saline (PBS) without Ca2+/Mg2+ using a blender (Braun Type 4184, Kronberg, Germany) and subsequent centrifugation at 3.000 × g for 5 min using a Heraeus Multifuge™ (Beckman Instruments GmbH Type 1 S-R, Vienna, Austria). The supernatant fluid containing blood components was discarded, pellets were resuspended in fresh PBS and centrifuged again (three times). Thereafter, the procedure was repeated three times with aqua dest.

Subsequently, 100 g wet weight of blood-free basal tissue were homogenized for 60 s in 100 mL Tris-NaCl buffer (50 mM Tris, 0.5 M NaCl, 4 mM EDTA, 2 mM N-Ethylmaleimide (NEM), pH 7.4) using the blender. Suspension was stirred overnight on a magnetic stirrer at 200 rpm and subsequently centrifuged at 7.000 × g for 15 min. Supernatants were collected and crystalline ammonium sulfate ([NH4]2SO4) was added to adjust for 30% final concentration. After 2 h of stirring, the extract was centrifuged at 7.000 × g for 15 min. Pellets were collected in 150 mL Tris-buffered saline (TBS) buffer and diafiltrated against 10× volumes of TBS. To precipitate collagen-4 contaminants, NaCl concentration was adjusted to 1.7 M by adding 150 mL of 3.4 M NaCl at a constant flow rate of 2 mL/min using a Minipuls Evolution® roller pump (Gilson Inc., Vienna, Austria) and stirred overnight at 200 rpm. Subsequently, the suspension was centrifuged at 7.000 × g for another 15 min. Supernatant containing native pLm-111 was either (a) diafiltrated against at least 3 volumes of TBS and stored at −80 °C (native pLm-111), or (b) diafiltrated against aqua dest. to remove residual salts, and concentrated to approximately 200 mL using TFF, and lyophilized (Christ Alpha 1-4 lyophilizer, Heraeus Schauer GmbH, Vienna, Austria). The resulting lyophilized pLm-111 was stored at −20 °C for up to 12 months before further use.

2.3 Biochemical Identification of pLm-111

2.3.1 Dot Blots

For native pLm-111 detection, dot blots were performed. 2 μL of either 1 mg/mL EHS laminin-111, pLm-111 (native or lyophilized), collagen-1 or recombinant laminin-111 from fibroblast cell culture (Sigma Aldrich, Vienna, Austria) were pipetted in duplicates on nitrocellulose membranes (Peqlab, Erlangen, Germany) and air-dried for 60 min. Thereafter, membranes were blocked with 5% skim milk powder in TBS buffer for 60 min and incubated with 1:2000 diluted monoclonal primary laminin-111 antibodies in TBS for another 60 min. After washing with TBS, membranes were incubated with peroxidase conjugated secondary antibodies (Abcam, CA, USA) for 60 min and signals were detected using a Multiimage Light Cabinet (BioZym, NY, USA).

2.3.2 SDS PAGE/Western Blot

SDS PAGE and western blot analysis were performed as previously described using the XCell SureLock™ Mini-Cell Electrophoresis System (Invitrogen, Vienna, Austria) [28, 29]. Briefly, 20 μg per lane of EHS laminin-111 (control), or lyophilized pLm-111 reconstituted in TBS buffer were resolved on 3–8% SDS-polyacrylamide gels (NuSep®, VWR, Austria), stained with 0.25% (w/v) Coomassie Brilliant Blue, or transferred onto nitrocellulose membranes (Peqlab) using the XCell II Blot Module (Invitrogen, Vienna, Austria). Membranes were blocked with 5% milk powder in TBS buffer containing 0.1% Tween (TBS/T) and incubated with anti-laminin-111 (polyconal 1:2000, AB11575, Abcam, USA) in 5% BSA-TBS/T at 4 °C overnight. Subsequently, membranes were incubated with peroxidase conjugated secondary antibodies (R1364HRP, Arctis GmbH, Germany) in 5% milk-TBS/T, and signals were detected using a Multiimage Light Cabinet (BioZym).

2.3.3 Amino Acid Analysis

Amino acid quantification was performed as previously described [30]. Briefly, pLm-111 was digested following a two-step protocol (enzymatical followed by chemical). 75 mg of lyophilized sample were incubated with 1 mL of 0.0125% protease from Streptomyces griseus in 1.2% TRIS/ 0.5% SDS pH 7.5 (adjusted with 0.1% HCl) solution for 72 h at 37 °C. Then 1 mL of 4% formic acid in ddH2O was added for chemical pre-digestion and the suspension was incubated for 2 h at 108 °C followed by lyophilization. The dried samples were reconstituted in 5 mL 0.6% TRIS and 7 M guanidine hydrochloride pH 8 for 2 h. After centrifugating (Sigma centrifuge, 3–18 K) the sample at 4800 rpm for 15 min at 4 °C, 1 mL of the supernatant was combined with 0.5 mL 4 M methansulfonic acid solution containing 0.2% tryptamine and incubated for 1 h at 160 °C. Subsequently, the solution was quantitatively transferred into a 5 mL volumetric flask, 225 μL 8 M NaOH and 0.25 mL internal standard were added and the flask was filled up with 2.2 M sodium acetate solution. The samples were then directly used for HPLC analysis.

A multi-amino acid standard mix was prepared by mixing the amino acid standard, a solution containing 2.5 mM each of asparagine, glutamine and tryptophan in MQ, a solution containing 2.5 mM each of taurine and hydroxyproline in 0.1 M HCl and a solution of the internal standards, i.e. 25 mM each of norvaline and sarcosine in 0.1 M HCl. Ten different concentrations of this standard mixture, ranging between 45 mg/L and 0.5 mg/L, were used for calibration.

The HPLC system Ultimate 3000 (Thermo Fisher Scientific, USA) was equipped with a pump (LPG-3400SD), a split-loop auto-sampler (WPS-3000 SplitLoop), a column oven (Col.Comp. TCC-3000SD) and a fluorescence detector (FLD-3400RS). Chromeleon 7.2 software was used for the control of the device as well as for the quantification of the peak areas. Chromatographic separation was achieved with a reversed phase column (Agilent Eclipse AAA, 3x 150 mM, 3.5 μm) a guard column (Agilent Eclipse AAA, 4.6 × 12.5 mM, 5 μm) and a gradient using eluent (A) 40 mM NaH2PO4 monohydrate pH 7.8 and eluent (B) MeOH/ACN/MQ (45/45/10, v/v/v). The protocol was run at a flowrate of 1.2 mL min−1, the column oven temperature was set to 40 °C and the injection volume was 10 μL. As most amino acids have no fluorophore in their structure, an in-needle derivatization step was performed using 0.4 M borate buffer, 5 mg/mL ortho-phthalaldehyde (OPA) in 0.4 M borate buffer containing 1% of 3-MPA, 2.5 mg/mL FMOC and 1 M acetic acid for pH adjustment. In order to guarantee sample quantification despite the derivatization step, every sample was spiked with 25 mM sarcosine in 0.1 M HCl and 25 mM sorvaline in 0.1 M HCl as internal standards. Primary amines and norvaline were detected at Ex 340 nm/Em 450 nm and secondary amines and sarcosine were detected at Ex 266 nm/Em 305 nm.

2.4 In Vitro Biocompatibility Testing of Isolated pLm-111

All in vitro experiments were performed with lyophilized pLm-111.

2.5 Adhesion

2.5.1 Primary Schwann Cell Isolation

All animals were euthanized according to established protocols, which were approved by the City Government of Vienna in accordance with the Austrian Law and the Guide for the Care and Use of Laboratory Animals as defined by the National Institute of Health.

Prior to Schwann cell isolation, sciatic nerves of adult male Sprague Dawley rats were dissected and kept in PBS on ice. Schwann cell isolation was performed as previously described [31], adapted from Kaekhaw et al. [32]. Cells were cultured in DMEM-D-valine (PAA, Austria), supplemented with 10% FCS, 2 mM L-Glutamine (PAA, Austria), 1% antibiotics (PAA, Austria), N2 supplement (Invitrogen, Germany), 10 μg/mL bovine pituitary extract and 5 μM forskolin.

2.5.2 Primary Schwann Cell Adhesion

For the Schwann cell culture, tissue culture plastic (TCP) was coated with poly-L-lysine and/or EHS laminin-111 or pLm-111. Briefly, 96-well plates were incubated with 0.01% (w/v) poly-L-lysine for 15 min at room temperature in a laminar flow-hood. Poly-L-lysine was removed and plates were left to dry for at least 2 h. Subsequently, wells were incubated with EHS laminin-111 or pLm-111 reconstituted in PBS (100 μg/mL) and incubated at 37 °C for 30 min. Laminin-111 solution was removed and plates were washed twice with PBS followed by UV sterilization.

Cell viability of Schwann cells on TCP, poly-L-lysin, EHS laminin-111, pLm-111 or on combinations of poly-L-Lysin with either EHS laminin-111 or pLm-111 was determined using MTT assay. Schwann cells, seeded at a density of 4 × 103cells/cm2 (n = 18), were incubated with culture medium containing 650 μg/mL MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] bromide for 1 h in a cell culture incubator (37 °C, 5% CO2 and 80% humidity). MTT reagent was discarded and MTT formazan precipitate was dissolved in 100 μL DMSO per well of a 96 well plate by shaking in dark for 20 min. Light absorbance at 550 nm was measured immediately and optical density (OD) values were corrected for an unspecific background on a microplate reader (Tecan Sunrise; Tecan Switzerland).

Proliferation of Schwann cells on TCP, poly-L-lysin (Lysin), EHS laminin-111, pLm-111 or on combinations of poly-L-Lysin with either EHS laminin-111 or pLm-111 was evaluated using a 5-bromo-2-deoxyuridine uptake assay (BrdU; Cell Proliferation ELISA assay Kit; Roche Diagnostics, Switzerland), according to manufacturer’s instructions. Briefly, 96-well plates of all groups were seeded with Schwann cells at a density of 4 × 103cells/cm2 (n = 18). Medium was changed to Schwann cell medium containing 100 μM BrdU and cells were incubated for 24 h at standard cell culture conditions (37 °C and 5% CO2). The culture plates were fixated with FixDenat® solution and incubated with anti-BrdU POD antibody solution for 45 min at room temperature. After washing the plate with PBS twice, substrate solution containing tetramethyl benzidine was added for 20 min. The reaction was stopped using 1 M H2SO4 and absorption was measured at 450 nm with 690 nm as reference wavelength on an automatic microplate reader (Tecan Sunrise; Tecan Switzerland).

2.6 NG 108-15 Outgrowth

NG 108–15 cell lines were purchased from ECACC (#88112302, Salisbury, U.K.) and cultured in DMEM high glucose supplemented with 10% FCS, 1% glutamine and 1% Pen/Strep.

24 well plates were incubated with 250 μL of EHS laminin-111 or pLm-111 at 100 μg/mL and UV sterilized for 30 min. Laminin solutions were removed and 12,000 cells were seeded (6000 cells/cm2, n = 12) on TCP, EHS laminin-111, or pLm-111 in medium supplemented with 20 ng/mL human beta neurotrophic growth factor β -NGF (Peprotech, Vienna, Austria) and incubated at 37 °C. Photographs were taken after 24, 48 and 72 h using an epifluorescence microscope (DMI6000B, Leica GmbH, Vienna, Austria). The neurite outgrowth was analyzed as previously described [33]. Briefly, microscopy pictures were processed in a blinded manner with Adobe Photoshop software by adjusting contrast/brightness. Then the neurite outgrowth was analyzed using AngioSys software (TCS Cellworks, London, UK). The obtained values were further statistically analyzed using Prism 5 (Graphpad, CA, USA).

2.6.1 Immunostaining

For actin/DAPI staining, the medium was aspirated and cells were washed with PBS before fixation in 4% formaldehyde for 10 min. The cells were washed three times with PBS, stained with Alexa Fluor 488 phalloidin (1:40) (Invitrogen) in the dark for 20 min, and washed two additional times with PBS. Then, DAPI staining (1:1000) for 5 min and two additional washing steps were performed before imaging on an epifluorescence microscope (DMI6000B, Leica GmbH, Vienna, Austria).

2.7 gfpHUVEC Network Formation

2.7.1 Human Umbilical Vein Endothelial Cells (HUVEC) Isolation

HUVEC were isolated from umbilical cords of healthy donors with the authorization of the local ethics committee of Upper Austria with written informed consent of the donors and according to established protocols as previously described [34, 35]. Cells (p6-p9) were cultured in EGM-2 medium (Lonza, Basel, Switzerland) supplemented with 5% FCS. Isolated HUVEC were retrovirally infected with expression vectors for fluorescent proteins using the Phoenix Ampho system as described elsewhere [36].

Network formation was investigated using a previously described vasculogenesis assay [37,38,39]. Briefly, 50 μL of pLm-111, EHS laminin-111, EHS collagen-4 or calf skin collagen-1 were pipetted per well in 96 well plates at two different concentrations of 500 μg/mL or 1 mg/mL, UV sterilized for 30 min and incubated at 37 °C for 2 h. Coating solutions were removed and 15.000 GFP-HUVECs were seeded (40.000 cells/cm2, n = 12) in 100 μL of EGM-2 medium. After 48 h of cultivation the networks were imaged and analyzed as previously described [33]. Fluorescence microscopic pictures of two independent experiments (different pLm-111 donors) were taken from two different fields per well and processed in a blinded way using Adobe Photoshop software (Adobe Systems, San Jose, USA) by adjusting contrast/brightness. Then, tube formation was analyzed using AngioSys software (TCS Cellworks, London, UK) and the AngioSys values were analyzed using Prism 5 (Graphpad).

3 Data Analysis

All experimental data is presented as mean ± standard deviation (SD) if not stated otherwise. Normal distribution of data was tested with the Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used to calculate statistical significance. For the NG108-15 outgrowth assay, a Two-Way ANOVA with Bonferroni post-test was used. P-values <0.05 were considered statistically significant. All calculations were performed using GraphPad software (GraphPad software, Inc., San Diego, CA, USA).

4 Results

4.1 Extraction of pLm-111 from Placenta

4.1.1 Yield and Purity

We have developed an effective method for isolating pLm-111 by extraction with a Tris-NaCl buffer combined with non-protein-denaturizing ammonium sulfate precipitation and rapid tangential flow filtration steps.

A detailed flow chart of the method is shown in Fig. 1.1. After defrosting, the chorionic membrane was removed and basal villous tissue was isolated. Major blood components were removed using PBS buffer/aqua dest. Thereafter, collagen remnants were removed and the residual laminin-111 diafiltrated against physiologic TBS buffer. The mean amount of pLm-111 after isolation was 175 ± 35 mg/100 g wet weight basal tissue (n = 7).

Fig. 1.1
figure 1

Graphical overview of steps required for the introduced rapid and efficient isolation of laminin-111 from human placenta (pLm-111). Full term placenta is dissected free of amnion/chorion followed by centrifugation to remove blood components, and further isolation/purification steps (salt-precipitation and diafiltration) to separate laminin-111. Final freeze-drying leads to powdery laminin-111 isolates

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By the use of Dot blot and monoclonal antibodies we assessed the presence of native laminin-111 in TBS buffer (Fig. 1.2a). After diafiltration and freeze-drying, laminin-111 is denatured and is therefore not detectable by the used monoclonal antibody (Fig. 1.2b). By using SDS-PAGE gels stained with Coomassie blue we assessed major protein bands in lyophilized pLm-111 (Fig. 1.2c) between 200 and 300 kDa. In western blot analysis using polyclonal antibodies, laminin-111 bands were clearly detected and matching with the major bands from the SDS-PAGE (Fig. 1.2d).

Fig. 1.2
figure 2

Isolated laminin-111 characterized by (a) Representative immunoblot of duplicates of 2 μg of purchased Engelbreth-Holmes Sarcoma (EHS) laminin-111, isolated native pLm-111 from two independent donors, purchased collagen-1 from rat tail against a monoclonal laminin-111 antibody. (b) Representative immunoblot showing duplicates of 2 μg of cell culture laminin-111, EHS laminin-111 or lyophilized pLm-111 from two independent donors against a polyclonal laminin-111 antibody. (c) Coomassie blue stained 3–8% SDS-polyacrylamide gel showing marker (M) (HiMark, Life Technologies), (1) EHS laminin-111 and (2, 3) two independent lyophilized pLm-111 isolates from two different donors. (d) Corresponding immunoblot showing 20 μg of (1) EHS laminin-111 and (2, 3) two independent lyophilized pLm-111 isolates from two independent donors loaded per lane and a primary antibody against polyclonal laminin-111

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Table 1.1 lists the amino acid composition of human laminin-111 α, β and γ-chains (www.uniprot.org), pLm-111 from three different donors and the amino-acid composition of EHS collagen-4 and human collagen-4 from placenta.

Table 1.1 Amino acid composition of pLm-111 (mean ± SD) from human placenta (Residues per 1.000 total amino acids residues)
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4.2 Biocompatibility of pLm-111

4.2.1 Schwann Cell Viability

The MTT assay was used to analyze Schwann cell viability on pLm-111 compared to EHS laminin-111. Cell viability on all three single coatings was significantly increased compared to the TCP control group (OD values: lysin 1536 ± 220, EHS laminin-111 1776 ± 195, pLm-111 1763 ± 216, TCP 503 ± 42, n = 18, Fig. 1.3a) but no significant difference among the three coating groups could be detected (p = 0.78). Schwann cells cultured on both combined coatings of lysin and EHS laminin-111 or lysine and pLm-111 showed increased viability (between 33% and 46%) compared to the single coatings or the TCP control. There were no significant differences between lysin/EHS laminin-111 and lysin/pLm-111 (OD values: lysin/EHS laminin-111 2285 ± 230, lysin/pLm-111 2362 ± 216, p = 0.75).

Fig. 1.3
figure 3

Schwann cell viability (MTT assay) and proliferation (BrdU assay) 24 h after seeding on tissue culture plastic (TCP) compared to EHS laminin-111 and pLm-111 coated wells (100 μg/mL), as well as poly-L-lysin/EHS laminin-111 and poly-L-lysin/pLm-111. Data is presented as mean + SD; significance tested with 1-way ANOVA followed by Tukey’s post test; *,** and *** indicates significant difference of p < 0.05, 0.01 and 0.005, respectively; n = 18

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The results of the proliferation analysis were similar to the viability assays. Schwann cells show higher proliferation on all three single coatings, compared to TCP (OD values: Lysin 3681 ± 512, EHS Laminin-111 3722 ± 470, pLm-1,113,822 ± 474, TCP 1871 ± 122, n = 18, Fig. 1.3b). No significant difference could be observed between the single coating groups (p = 0.87). Proliferation on combined coating of lysine and EHS laminin-111 or lysine and pLm-111 resulted in increased proliferation (between 23% and 33%) compared to the single coatings or the TCP but no differences between EHS laminin-111 and pLm-111 were detectable (OD values: EHS Laminin-111 4938 ± 297, pLm-111 5034 ± 381, p = 0.79).

4.2.2 NG 108-15 Outgrowth

An outgrowth assay was used to analyze NG 108–15 cells on pLm-111 compared with EHS laminin-111 (Fig. 1.4). After 24 h, the total neurite outgrowth (TCP 86 ± 3 μm, EHS laminin-111,268 ± 13 μm, pLm-111,519 ± 16 μm, n = 12) and the number of tubules (TCP 5 ± 1, EHS laminin-111 11 ± 3, pLm-111 20 ± 2, n = 12) on pLm-111 were significantly increased compared to the TCP control and EHS laminin-111, but no significant difference between EHS laminin-111 and TCP could be detected. After 48 h, the total neurite outgrowth (TCP 71 ± 6 μm, EHS laminin-111,590 ± 110 μm, pLm-111,848 ± 240 μm, n = 12) and the number of tubules (TCP 3 ± 1, EHS laminin-111 21 ± 5, pLm-111 33 ± 9, n = 12) on both coatings were significantly increased compared to the TCP control, but no significant difference between EHS laminin-111 and pLm-111 could be detected. After 72 h, the total neurite outgrowth (TCP 96.9 ± 1 μm, EHS laminin-111,382 ± 4 μm, pLm-111 1024 ± 6 μm, n = 12) and the number of tubules (TCP 6 ± 1, EHS laminin-111 20 ± 4, pLm-111 47 ± 5, n = 12) on pLm-111 were significantly increased compared to EHS laminin-111.

Fig. 1.4
figure 4

Upper panel: fluorescence micrographs of phalloidin (green) and DAPI (blue) stained NG 108-15 cells cultivated on tissue culture plastic (TCP), EHS laminin-111 or pLm-111 at concentrations of 100 μg/mL; Scale bars = 100 μm; Lower panel: analysis of the neurite outgrowth: total neural length and number of tubules per field of view of NG108 cells after 24, 48 and 72 h of cultivation. Data is presented as mean + SD; significance tested with 2-way ANOVA followed by Bonferroni post test; *,** and *** indicate significant difference of p < 0.05, 0.01 and 0.005, respectively; n = 12

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4.2.3 HUVEC Network Formation

A well-established vasculogenesis assay was used to analyze gfpHUVEC with fully supplemented EGM-2 medium on pLm-111 from two independent isolations (donors D1; D2) and compared with EHS laminin-111 (Fig. 1.5). After 24 h, a cell network was formed on EHS laminin-111 and pLm-111 at 1 mg/mL but neither on EHS collagen-4 nore on lower pLm-111 coating concentrations or TCP. After 48 h, the networks were analyzed. There was no significant difference between the number of tubules (EHS laminin-111,134 ± 17, pLm-111 D1 124 ± 13; D2 157 ± 18, n = 12) or the total tubule length (EHS laminin-111 35 ± 6 mM, pLm-111 D1 37 ± 2; D2 42 ± 5 mM, p < 0.5, n = 12) between both laminin-111 coatings. The mean tubule length (EHS laminin-111,240 ± 7 μm, pLm-111 D1 315 ± 17; D2 300 ± 13 μm, n = 12) and the number of junctions (EHS laminin-111,284 ± 28, pLm-111 D1 410 ± 42; D2 463 ± 47, n = 12) were significantly increased on pLm-111 compared to EHS laminin-111.

Fig. 1.5
figure 5

pLm-111 at higher concentrations promotes vasculogenesis using gfpHUVEC (a) Fluorescence micrographs of gfpHUVEC 48 h after seeding on EHS laminin-111, pLm-111, or EHS collagen-4 at concentrations of 1 mg/mL. Scale bar = 400 μm. (b) Network characteristics (total/mean tube length, number of junctions/tubules) of gfpHUVEC, seeded on EHS laminin-111 or pLm-111 from two independent donors (pLm-111 D1; D2). Data is presented as mean + SD; significance tested with 1-way ANOVA followed by Tukey’s post test; *,** and *** indicates significant difference of p < 0.05, 0.01 and 0.005, respectively; n = 12

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5 Discussion

Although laminin-111 from EHS tissue was already described more than 30 years ago, about 20,000 laminin publications in 2016 proved the ongoing interest in this key protein [2]. Laminin-111 is naturally present during embryonic development and has been shown to be a useful biomaterial for the cultivation of stem cells [7, 26, 40, 41]. Beside, it has been used for various applications in tissue engineering and regenerative medicine, eg. cultivation of neuronal cells [7, 11], angiogenesis studies [5, 13], or wound healing studies [6, 15]. A robust method for culturing human pluripotent stem cells under xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine [42]. Especially in the research of muscle tissue biology, laminin-111 has been shown to improve skeletal muscle stem cell quality and function [43]. In this regard, Goudenne et al. could demonstrate that intramuscular injection of laminin-111 increased muscle strength and resistance in mice and could potentially be used to treat Duchenne muscular dystrophy [44].

Regarding clinical applicability, caution must be taken regarding the tissue origin of the extracted biomaterials. Proteins extracted from xenogenic tumor tissues are not suitable for clinical applications. Non-human proteins are reported to provoke immune responses in patients [45], and carry the risk of xenogenic disease transmission [46, 47]. Therefore, human sources are regarded as the best option for the generation of medicinal products [30]. In this regard, human placenta is a highly vascularized organ [48], and it therefore harbors high amounts of basal membrane proteins [49]. Human placenta tissue is available in sufficient amounts and consistent quality for large industrial scale processes. Moreover, it has been shown to exhibit excellent anti-inflammatory and antibacterial properties, [50] which has been favoring its use to treat non-healing wounds for decades [51,52,53].

Commercial success of a medicinal product is dependent on the balance of efficacy and cost-effectivness [54]. Several studies have been performed to isolate laminin isoforms from placenta using salt precipitation or pepsin digestion and chromatography [20,21,22,23,24]. However, the protocols to isolate laminin-111 described in the literature to date are time consuming, work intensive and show low extraction efficiency. By modifying the protocols described in literature, we were able to (1) significantly increase the extraction efficiency of laminin-111 yield from placenta tissue compared to reported isolation protocols and (2) to shorten the isolation time from almost 2 weeks to a total of only 4 processing days (Fig. 1.1) [20,21,22]. In our protocols, tangential flow filtration instead of dialysis was used since it was less time-consuming and further allows easy and rapid concentration of laminin isolates. Collagen-4 remnants were removed by precipitation with 1.7 M NaCl [18].

Native laminin-111 from human placenta was assessed using Dot Blots. Residual salts from TBS buffer were removed by tangential flow filtration against aqua dest and the proteins were freeze-dried to make process ability and storage easier. On the average, 175 ± 35 mg of laminin-111 was isolated from 100 g of placenta tissue using the isolation protocol described here. In western blot analysis for laminin-111 and SDS-PAGE analysis, two bands around 200 kDa were clearly visible, which represent the α1 and ß1 chains, as described in literature [1, 19, 55]. Interestingly, a third protein band was visible with a molecular weight of approximately 300 kDa in both the pLm-111 isolates and in EHS laminin used as control. This high molecular weight band most likely represents the reduced γ-chain, which usually appears around 400 kDa [2, 19]. The HPLC- based amino acid quantification analysis is consistent with the data from www.uniprot.com, however low amounts of hydroxyproline were still detectable. Most probably, these could be attributed to impurities of the isolate with collagen-4, another major component of BMs beside laminin [55].

Freeze-dried pLm-111 was used as a coating substrate and compared to xenogenic standard proteins using Schwann cells. These experiments indicated that the pLm-111 isolates show bioactivity comparable to commercially available xenogenic proteins. Furthermore, pLm-111 promoted rapid attachment of various cells including Schwann cells, HUVEC or NIH3T3 fibroblasts (data not shown). NG 108-15 cells rapidly developed a complex neurite outgrowth on pLm-111 with at least similar effectiveness compared to xenogenic proteins.

In addition, laminin-111 is the principal factor for endothelial cells to differentiate into interconnected tubules on Matrigel, a laminin-111-rich (around 70%) gel, extracted from basement membrane tumor materials from mice [56]. However, beside its tumorigenic origin, Matrigel is a heterogenic mixture of various extracellular matrix proteins and pro-angiogenic growth factors. For potential clinical applications, a single protein would be an advantageous material. EHS laminin-111 at concentrations of 1 mg/mL promotes the differentiation of gfpHUVEC into interconnected networks, but so does pLm-111 from human origin with at least similar performance (total/mean tube length, number of tubules/junctions).

The 2D cell culture experiments described above have been performed with freeze-dried laminin-111 stored at −20 °C for at least 6 months. Moreover, in other in vitro experiments we have used the isolated lyophilized pLm-111 materials after storing them for up to 18 months without noticing alterations in stability or activity (data not shown).

To conclude, we could establish a simple, rapid and effective method to isolate laminin-111 from human placenta. This pLm-111 clearly demonstrates its applicability as a biomaterial of human origin with strong bioactive potential for a broad spectrum of in vitro and potentially in vivo tissue engineering approaches.

6 Conclusion

Summarizing, we established an effective method for isolating laminin-111 from human placenta, a clinical waste material. Laminins are routinely used in cell culture to on the one hand improve attachment of cells (eg Schwann cells), and on the other hand induce specific functions (eg HUVEC). The availability of this potent and versatile protein offers new, fully human, approaches for neuronal and endothelial in vitro tissue engineering and may provide a new platform technology for clinical use with an increased overall safety profile for patients.