Measuring the Confluence of iPSCs Using an Automated Imaging System

Summary

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

Abstract

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

Introduction

Induced pluripotent stem cells (iPSCs) are obtained from somatic cells and can be differentiated into different cell types. They are often used as a system to model disease pathogenesis or perform drug screening, and also offer the potential to be used in the context of personalized medicine. Since iPSCs have great potential, it is important to fully characterize them for use as a reliable model system. We previously showed the importance of growing iPSCs in a hypoxic environment as these cells rely on glycolysis and an aerobic environment can cause redox imbalance¹. iPSCs are also vulnerable to other culture conditions, particularly the extracellular environment. Optimization of culture conditions is a key issue to keep them healthy and proliferating. A healthy iPSC culture will lead to healthy differentiated cells that generally are the endpoint of the model used to understand molecular, cellular and functional features of specific human disorders or cellular processes.

In this study, a simple protocol has been used to test the confluence of iPSCs using different coating conditions in separate wells. iPSCs require a feeder layer of murine embryonic fibroblasts (MEF) in order to properly attach, but the coexistence of iPSCs and MEF makes it difficult to perform analysis like RNA or protein extraction since two populations of cells are present. In order to avoid the feeder layer, different proteins belonging to the extracellular matrix (ECM) have been used to recreate the natural cell niche and to have feeder free iPSC culture. In particular, Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which is enriched in extracellular matrix proteins (i.e., laminin, collagen IV, heparan sulfate proteoglycans, entactin/nidogen, and growth factors)^2,3. The other used coating conditions are instead purified proteins with known relevance in building the ECMs: laminin-521 is known to be secreted by human pluripotent stem cells (hPSCs) in the inner cell mass of the embryo and it is one of the most common laminins in the body after birth^{4,5,6,7,8,9,10,11}; vitronectin is a xeno-free cell culture matrix known to support growth and differentiation of hPSC^{12,13,14,15,16}; fibronectin is an ECM protein important for vertebrate development and the attachment and maintenance of embryonic stem cells in a pluripotent state^{17,18,19,20,21,22,23,24,25}. Since different coating conditions are available, we compare them in terms of their effect on iPSCs’ confluence.

Protocol

1. Coating 96 well plates

NOTE: Different coatings were tested in the same plate but separate wells (see Supplemental File).

1. Dilute the Matrigel 1:100 in DMEM. Add 100 µL per well to the 96 well plates and incubate for 1 h at room temperature. Following this, remove the solution and wash the wells with 100  µL of DMEM twice.
2. Dilute laminin (20 µg/mL, LN-521) in PBS (with calcium and magnesium). Add 100 µL to the well and incubate at 4 °C overnight. The following day perform two washes with DMEM before seeding the cells.
3. Dilute vitronectin (10 µg/mL) in dilution buffer. Add 100 µL per well to the 96 well plate and incubate for 1 hour at room temperature. Wash the wells with PBS (without calcium and magnesium) before plating the cells.
4. Dilute HU-Fibronectin (30 µg/mL) in ddH₂O. Add 100 µL to the wells and incubate at room temperature for 45 minutes. Following this, wash the wells with the medium before seeding the cells.

2. Maintenance of iPSCs in culture

NOTE: iPSCs were purchased commercially. The iPSCs were derived from healthy human fibroblasts and reprogrammed using episomal technology.

1. From the -80 °C freezer or liquid nitrogen, thaw the cryopreserved iPSCs in a 37 °C water bath. Clean the vial containing the cells with 70% ethanol prior to moving it into the biological safety cabinet.
2. Add the cell suspension to 5 mL of pre-warmed cell culture media (e.g., mTeSR1) drop by drop with a 1000 µL pipette in a 15 mL sterile conical tube.
3. Centrifuge the cells at 304 x g for 5 min at room temperature (RT).
4. Remove the media and resuspend the cell pellet in 4 mL of cell culture medium.
5. Plate the cell suspension into two wells of the 6 well plates (10⁵ iPSCs per 6-well cell culture dish), where the mouse embryonic fibroblasts (MEFs) have been plated previously. Seed MEFs two days before plating iPSCs at a density of 2.4 x 10⁴/cm² in DMEM (containing 10% Fetal Bovine Serum, 1% L-glutamine and 1% Penicillin-Streptomycin).
6. After seeding, supplement the cell media with 10 µM of ROCK inhibitor Y-27632.
7. Grow the iPSCs on MEFs for the first 4-5 weeks and then in feeder free condition (MEF free condition), using one of the coating of interest (see step 1 and Table 1) in mTeSR1.
8. When the iPSCs are 70-80% confluent, passage 1:4 using 0.5 mM EDTA treatment for 3-5 min at RT. Add 1 mL of 0.5 mM EDTA for a 6 well plate (or proportional quantities for other types of plates). Transfer to new wells in feeder-free conditions and incubate at 37 °C, 5% CO2, 20% O₂.  
9. Change the media with fresh mTeSR1 every day and split the cells every 2 days.

3. Characterization of cell confluence

1. Use 96 well plates for the experiments.
2. Seed 10,000 cells per well following at least 1 month of culturing in feeder free condition in order to be sure that the MEFs were not passaged. Use disposable counting slides  to count the cells with the optical microscope.
3. Perform the experiments in triplicate. Therefore, test each seeding condition in three wells.
4. Perform automated image acquisition from day 1 following seeding using a cytometer in bright-field mode. Perform automated image acquisition every 24 h for 5 days. For detailed information on the experimental parameters, refer to the Supplemental File.
5. Use auto-contrast and auto-exposure to better visualize cells.
6. Set the analysis setting (see Supplemental File) for confluence analysis to apply a mask of 60%, 80% and 100% per well, in order to evaluate the changes in focus due to the light refraction at the border of the wells. Use the different mask analysis setting mentioned above to analyze the cell confluence at each time point.

4. Statistical analyses

1. Report quantitative results as means ± standard error of the mean (SEM).
2. For comparing overall differences of the different coating conditions, obtain data using the same samples and perform the Student’s paired-sample t-test. P values less than 0.05 are considered statistically significant, and all reported p-values are two sided.

5. Characterization of the cytoskeletal microfilaments

1. Fix cells with 4% paraformaldehyde (4% PFA) in PBS for 10 min at RT, followed by two washes in PBS (10 min total).
2. Add 100 µL of blocking solution (composed by 5% BSA, 0.1% Triton in PBS) to each well for 1 h at RT.
3. Remove the blocking solution and wash the samples twice with PBS for 10 min.
4. Add 100 µL of the phalloidin-conjugate working solution per sample and incubate for 1 h at RT.
5. Wash cells twice with PBS (10 min at RT).
6. Stain nuclei with Hoechst 33342 diluted 1:10000 in PBS for 10 min at RT.
7. Remove the Hoechst solution and wash the cells twice with PBS for 10 min each time.
8. Wash the sample with H₂O and let dry under a chemical hood.
9. Add 100 µL of mounting media (i.e. PBS:glycerol, 1:1) to cover the cells and preserve fluorescence of samples.
10. Observe cell at Ex/Em 493/517 nm on a laser-scanning confocal microscope equipped with a white light laser (WLL) source and a 405 nm diode laser. Acquire sequential confocal images using a HC PLAPO 40x oil-immersion objective. Use the same laser power, beam splitters, filter settings, pinhole diameters and scan mode for all examined samples.

Results

In this study, we investigated iPSCs confluence when grown on different coating conditions. Using a cytometer, we were able to obtain readily informative results in triplicates in 5 days. Since iPSCs hardly attach to plastic vessels and a coating is necessary to support their proliferation, we decided to monitor the confluence of human iPSCs as it is indicative of the health of the cell culture and it may reflect on their differentiation potential. After in vitro expansion, we seeded the iPSCs on different ECM substrates...

Discussion

The use of iPSCs for disease modeling and future drug screening together with their possible application in precision medicine makes it a technology of great relevance and for this reason we believe that it is necessary to clearly understand the in vitro culturing condition that better resemble the physiological situation of embryonic stem cells. In this context, we tested different ECM coatings using wild type iPSCs in order to understand the conditions that allow the cells to remain in a healthy and undifferentiat...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile	Corning	4488	Tool
15 mL high-clarity polypropylene (PP) conical centrifuge tubes	Falcon	352097	Tool
1x PBS (With Ca2+; Mg2+)	Thermofisher	14040133	Medium
1x PBS (without Ca2+; Mg2+)	Euroclone	ECB4004L	Medium
5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile	Corning	4487	Tool
Cell culture microplate, 96 WELL, PS, F-Bottom	Greiner Bio One	655090	Support
Cell culture plate, 6 well	Costar	3516	Support
DMEM (Dulbecco's Modified Eagle's Medium- high glucose)	Sigma	D5671	Medium
EDTA	Sigma	ED4SS-500g	Reagent
Epi Episomal iPSC Reprogramming Kit	Invitrogen	A15960	Reagent
FAST - READ 102	Biosigma	BVS100	Tool
Fetal Bovine Serum (FBS)	Gibco	10270106	Medium
Fibronectin	Merck	FC010	Coating
Glycerol	Sigma	G5516	Reagent
H2O		MILLIQ
Hoechst	Thermofisher	33342	Reagent
Laminin 521	Stem Cell Technologies	77003	Coating
L-Glutamine (200 mM)	Gibco	LS25030081	Reagent
Matrigel	Corning Matrigel hESC-Qualified Matrix	354277	Coating
Mouse embryonic fibroblasts (MEF)	Life Technologies	A24903	Coating
MTESR1 Medium	Stem Cell Technologies	85851	Medium
MTESR1 Supplement	Stem Cell Technologies	85852	Medium
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122	Reagent
Phalloidin	Sigma	P1951	Reagent
Vitronectin	Stem Cell Technologies	7180	Coating
Y-27632	Sigma	Y0503	Reagent