Page 54 - Power of Stem Cells- arthritis and regeneration

Theranostics 2018, Vol. 8, Issue 4 908

medium (α-MEM) with l-glutamine from Quantitative real-time PCR
Macopharma supplemented with 5% platelet lysate Total RNA was isolated from the cells using
and 3.3 IU/mL heparin up to passage 3 (7). TRIzol (Invitrogen) according to the manufacturer’s
Characterization of MSCs fits the internationally instructions. Total RNA (200-500 ng) was reverse
convened minimal criteria for these cells (41). The transcribed using the High-Capacity cDNA Reverse
ATMP MSCs used for extracellular vesicles Transcription Kit (Applied Biosystems). Real-time
production in this study were obtained from surplus polymerase chain reactions (PCRs) were performed
cells of one male and one female donor used for the using FastStart Universal SYBR Green Master mix
IMPACT trial (NCT02037204) (7) and cultured for (Roche Diagnostics) in a LightCycler 96 (Roche
additional passages (passage 4-7) in α-MEM (Gibco Diagnostics) according to the manufacturer’s
Invitrogen, Carlsbad, CA, USA) supplemented with instructions. Quantification was performed relative to
5% human platelet lysate (PL), 100 U/mL penicillin the levels of the housekeeping gene 18S and
and 100 μg/mL streptomycin (Gibco Invitrogen) and normalized to control conditions. The data analysis
10 U/mL heparin and maintained at 37 ºC and 5% was performed using the 2−ΔΔCT method (25). The
CO 2. The PL was depleted from PL-derived primer sequences are listed in the table in
extracellular vesicles by overnight centrifugation at Supplemental Material and Methods. The amplified
100,000 × g. PCR fragment extended over at least one exon border
In vitro cartilage regeneration assay (except for 18S).
Cells were cultured in fibrin glue constructs. Per Proliferation
donor the following was performed: For the fibrin To test for proliferation in monolayer cultures, 10
glue constructs, passage two chondrocytes were μM 5-ethylnyl-2’-deoxyuridine (EdU, Invitrogen) was
resuspended in a 1:15 diluted fibrinogen component supplemented to the culture medium. After 5 days,
of Beriplast (Baxter) using PBS and 50 µL was injected medium was aspirated, cells were washed with PBS,
in a 96-well plate and combined with a 1:50 diluted formalin fixed for 15 min and permeabilized. EdU
component of thrombin, resulting in 100 µL constructs was visualized using Click-iT EdU Alexa Fluor 488
containing 0.25 x 10 cells per construct. Chondrocytes Imaging kit (Invitrogen) according to the
6
were subsequently cultured in 24-well plate in DMEM manufacturer’s instructions. Cells were photographed
supplemented with 2% insulin–transferrin–selenium using an EVOS FLoid Cell Imaging microscope and
(ITS)-X (Invitrogen), 2% ASAP, 2% human serum analyzed in ImageJ.
albumin (Sanquin Blood Supply Foundation), and 1% For fibrin glue constructs in regeneration
penicillin/streptomycin (100 U/mL, 100 mg/mL). cultures, an AlamarBlue assay (10% (w/v) resazurin

In vitro inflammation assay (Alfa Aesar, Thermo Scientific) was performed to
Chondrocytes were cultured in monolayer at measure metabolic activity according to
manufacturer’s protocol.
37°C and 5% CO 2 at a seeding density of 25,000
cells/cm in medium consisting of DMEM Collagenase activity assay
2
supplemented with 10% FBS, 100 U/mL penicillin, To analyze collagenase activity, the Enzchek
and 100 mg/mL streptomycin. After pre-incubation Gelatinase/Collagenase Assay Kit (Invitrogen) was
for 24 h, cells were treated with 10 ng/mL TNF-alpha used according to the manufacturer’s instructions. DQ
(Immunotools). Collagen Fluorescein conjugate was added to
Glycosaminoglycan analysis undiluted conditioned medium. This was incubated
for 4 h at ambient temperature, protected from light.
After 4 weeks of culture, samples were digested The fluorescence was measured at 480 nm.
overnight in a papain digestion buffer (250 mg/mL
papain (Sigma-Aldrich), 0.2 M NaH2PO4, 0.1 M Histological analysis
EDTA, 0.01 M cysteine) at 60°C before quantification Samples were dehydrated using graded alcohol
of the sulphated glycosaminoglycans (GAG) content steps, immersed in xylene, embedded in paraffin, and
with a 1,9-dimethylmethylene blue (DMMB) assay. cut into 5 mm sections. To evaluate the proteoglycan
The absorption ratio was set at 540–595 nm using content, 0.125% safranin-O (Merck counterstained
chondroitin-6-sulfate (Sigma-Aldrich) as a standard. with Weigert’s hematoxylin [Klinipath], 0.4% fast
DNA content in the papain digests was determined green [Merck]) staining was used. Type II collagen
using a Picogreen DNA assay (Invitrogen) according deposition was determined by immunohisto-
to the manufacturer’s instructions. chemistry. Antigen retrieval was performed by
subjecting the sections to 1 mg/mL pronase

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