Suchitra Derebail, Casthri Krishnamurthy, Ong Hong Boon, Ang Kailin, Nur Amilia Bte M Isa, Nur Ayuni Bte Hassan Jaya, Orr Hui Min.
Republic Polytechnic, 9 Woodlands Ave 9, Singapore 738964
Embryonic stem cells (ES) are unique cells obtained from structures called blastocysts or early mammalian embryos. In the case of mice, blastocysts are formed around day 4 - 4.25 during embryogenesis. They consist of a special group of cells called the ICM or Inner Cell mass, an outer layer called trophoblast and a cavity or the blastocoele. ES cells were first isolated from the ICM in 1981, and this method is still in use1-2. ES cells have two unique features; 1. They are unspecialized and have the potential to self-renew infinitely. 2. They can undergo spontaneous or induced differentiation under experimental conditions, into any specialized cell type, for example, cardiac muscle cells, neurons of the brain etc. These features have infused ES cells with tremendous potential to repair and replace organs. ES cells are typically cultured as colonies of adherent cells, in the presence of LIF or leukemia inhibitory factor. They are either cultured in gelatin-coated culture dishes or as an overlay on feeder cells. LIF works through an intricate pathway that involves a two-part receptor complex3. When LIF binds to this receptor, it activates a latent transcription factor; STAT3. The events that follow ensure that ES cells proliferate without loss of pluripotency or stemness. In vitro differentiation of ES cells into specialized cell types is usually preceded by the formation of three-dimensional aggregates called embryoid bodies (EBs)4. These aggregates are grown in suspension without addition of LIF, and in the absence of feeder layers. Under such in vitro conditions, EBs appear to simulate the events of in vivo early embryogenesis. From this stage onwards, EBs are able to spontaneously differentiate into the three germ layers of the embryo; ectoderm, mesoderm and endoderm5. At this stage, the addition of growth factors has been shown to induce EBs to differentiate into specific cell types. For example, addition of fibroblast growth factor and bone morphogenetic protein can induce EBs to develop into cardiomyocytes6. Other growth factors like hepatocyte growth factor, retinoic acid (RA) and epidermal growth factor (EGF) have been extensively used in differentiation studies.
Researchers have developed many methods to develop EBs and have successfully differentiated them into numerous cell types; fibroblasts, cardiomyocytes, chondrocytes, insulin-producing cells etc. The quality of EBs is a crucial factor for successful differentiation7. The two common methods of generating EBs on a small scale are; 1. Suspension cultures. 2. Hanging drop cultures. Each method has its own advantages and disadvantages (See Figure 1). Cell culture companies offer a limited assortment of products for the generation of uniform EBs. For example, AggreWell plates from Stemcell technologies contain microwells with a diameter of 400 μm, which force ES cells into uniform-sized EBs8-9. This product promises an easy and standardized approach to the production of highly uniform EBs. Another related product is StemPro EZpassage from Life technologies10. It is a sterile, disposable, passaging tool. It is designed to assist in cutting ES colonies into uniform sized pieces for passaging, with the potential to be developed into a tool for generating uniform sized EBs (See Figure 2).
In this project conducted in Republic Polytechnic for a period of six months, four different methods, as mentioned above, for the generation of uniform EBs from mouse embryonic stem cells were evaluated, in terms of ease-of-use, time taken, pricing and the quality of EBs. In each case, EBs generated were also assessed for their ability to differentiate spontaneously into cardiomyocytes. The methodology used and the results obtained are discussed in separate sections.
All the cell culture related work described below was conducted in a biosafety cabinet (Class II). The cells were incubated in a 37°C incubator with 5% CO2.
- Suspension cultures - This method was originally developed by Doetschman et al. using bacteriological plates. After thawing from an initial vial of cells, mouse ES cells were sub-cultured several times on gelatin-coated culture dishes, in LIF containing complete growth media. On day 2 after subculturing, ES colonies were dissociated from each other, enzymatically, using trypsin enzyme. The cells were first washed with buffer solution, trypsin enzyme was then added and the cells were incubated for 2-3 minutes in the incubator. After neutralizing the enzyme with appropriate volume of culture media and centrifugation, small clumps of ES colonies were then transferred into 60 mm bacteriological dishes containing 10 ml of complete growth media without LIF. These dishes are devoid of the special cell culture treatment that causes ES colonies to adhere to plastic surfaces. If left undisturbed, the ES clumps tend to aggregate naturally and remain in suspension. On days 4-5 after sub-culturing, the EBs were large enough for differentiation assays (See Figure 1).
- Hanging drops – The initial steps of this method are the same as those in suspension cultures, described above. On day 2 of the culturing process, ES cells were enzymatically dissociated and a cell count was obtained using a haemocytometer. The cell suspension was diluted to get a concentration of 2.5 X 104 cells/ml. 20μml droplets of 500 cells per droplet were plated in rows, under the lid of a bacteriological dish. The lid was then carefully placed over the bottom of the bacteriological dish. About 15 ml of serum-free growth media was placed in the bottom of the dish, to provide adequate hydration to the hanging drops. These plates were left undisturbed in the incubator for another two days or until the EBs reached a suitable size. They were then transferred into bacteriological plates as suspension cultures and allowed to grow for another two to three days. On days 6 – 7 after sub-culturing, the EBs were usually apt for differentiation (See Figure 1).
- AggreWell plates – These are sterile, cell culture grade plates provided by Stemcell Technologies. They contain microwells that help in generation of EBs (See Figure 2). The steps in this section are extracted from manufacturer's instructions in Technical manual – Reproducible and uniform embryoid bodies using AggreWell plates, Version 3.0.0, catalog number 27845. The plates were removed from their packaging in the sterile environment of the biosafety cabinet. About 0.5 ml of culture media was added into each well and the plate was centrifuged for 5 minutes at 100 X g, on a swinging bucket rotor. This step ensures the removal of air bubbles from the microwells. After counting the cells from a day 2 culture, ES cells were diluted to a concentration of 6 X 104 cells/ml. As shown in Table 1 below, this concentration would yield EBs containing 500 cells each. Alternatively, any other concentration could be chosen according to the needs of the experiments. One ml of cell suspension was added to each well, without aspirating the previously added volume of media. The total volume was adjusted to 2 ml, the cells were gently pipetted several times to distribute them equally and the plate was then centrifuged again for 3 minutes at 100 X g. The plates were incubated for 2 days, the EBs were gently transferred to bacteriological plates as suspension cultures and allowed to grow for an additional two days. On day 7 after sub-culturing, EBs were large enough for use in differentiation assays.
- StemPro EZpassage (Some of the steps in this section are adapted from the product insert – StemPro EZpassage disposable stem cell passaging tool, catalog number 23181-010.)
The tool has a miniature roller blade attached to a handle. When the blade is applied in a rolling fashion, with constant and uniform pressure over the cells, uniform-sized ES colonies can be obtained. Mouse ES cells were cultured for two days. In the process of subculturing, the cells were washed with buffer solution and treated with trypsin enzyme. The cells were kept at room temperature instead of transferring to the incubator. Enzyme treatment was limited to one minute. The passaging tool was removed from the packaging under a biosafety cabinet. The tool was rolled over the cells in the plate, as shown in Figure 2. After rolling the tool over all the cells in one direction, the culture plate was rotated 90 degrees and the tool was rolled over the cells again. The cut colonies were then gently resuspended and transferred to bacteriological plates for growth in a suspended state. On days 4-5 after sub-culturing, the EBs were large enough for differentiation assays.
Differentiation assays – The EBs obtained in the four methods were used separately, in downstream differentiation assays. Five to six EBs were plated into each well of a gelatin-coated 6-well plate and returned to the incubator. Cells were observed every day to check for the adherence of EBs and differentiation into various cell types, including cardiomyocytes. Culture media was changed on alternate days using complete culture media without any growth factors or LIF. On days 9-10, several patches of beating or contracting cardiomyocytes were observed in dishes containing EBs generated using hanging drops and AggreWell plates. Table 2 shown below lists the parameters compared using the four methods.
In this study, four methods for the generation of uniform embryoid bodies were conducted and evaluated using a set of parameters; ease-of-use, cost, quality of EBs, differentiation into cardiomyocytes, and time taken to obtain good quality EBs for differentiation assays (Figure 3). Suspension cultures and hanging drop methods are conventional ones that have been in use for a long time. AggreWell plates and StemPro EZ passage tools are products offered by Stemcell technologies and Life Technologies, respectively. Each method was observed to have some advantages and disadvantages (see Table 2).
In suspension cultures, EBs are generated from day 2 ES cells after trypsinization. This is a fast, easy and inexpensive method. However, since the number of cells is not determined before making EBs, generally variable sized EBs were observed. The EBs failed to develop into contracting cardiomyocytes during the differentiation process. In contrast, hanging drops are made after determining the exact cell count. This method is easy to control and the sizes of EBs can be pre-determined by diluting the cell suspensions to give 500 cells per droplet. This method lends itself to easy manipulations by the user. Inside the hanging drops, the cells tend to aggregate at the bottom of the drop due to surface tension. If left undisturbed in the incubator, EBs are ready to be grown in suspension within two days. However, the major downsides of this method are that it is time-consuming and labour-intensive. This method adds 2-3 days to the overall process of EB formation. Other minor disadvantages are that it is not possible to change media in the drops or observe them under a microscope. Additionally, pipetting errors could cause changes in droplet volume and result in heterogenous EBs.
The AggreWell plates resemble a regular 24-well culture plate, except for the central 8-wells that consist of microwells with a diameter of 400μm. Cells when placed in the microwells and centrifuged are forced to aggregate into uniform-sized EBs by the walls of the microwells. About 80% of the microwells contain remarkably identical EBs. Moreover, these EBs were able to develop successfully into contracting cardiomyocytes. However there were some missing EBs or abnormal-sized EBs in about 20% of the wells. The major disadvantages of this system are the cost and the requirement of gentle pipetting skills to disperse the cells equally in all wells. The StemPro EZ passage tool was originally designed to obtain uniform-sized clumps of ES cells for the purpose of subculturing. But they were tested for their potential to generate uniform EBs. Similar to suspension cultures, this tool is fast and easy to use. However, the EBs generated were highly variable in size and tended to fall apart very quickly. When plated, the EBs failed to develop into cardiomyocytes.
Overall, the project findings reaffirm the use of hanging drops as the most reliable way to generate uniform-sized EBs, in spite of the additional steps involved. AggreWell plates are a good, albeit, expensive alternative to hanging drops. The quality of EBs in both these methods is comparable and they are efficient when utilized in differentiation assays for cardiomyocytes.
The quality of embryoid bodies (EBs) derived from pluripotent embryonic stem cells plays a crucial role in successful differentiation of ES cells into cells of the three germ layers; ectoderm, mesoderm and endoderm. There are a number of drawbacks in the traditional methods of EB formation; suspension cultures and hanging drops. While the former does not allow for controlled EB formation, the latter is a time-consuming and laborious process. Both techniques fail in controlling the microenvironment of the ES cell. During differentiation, ES cells are regulated by many cues from the microenvironment via cell-cell, cell-extracellular matrix or cell-growth factor interactions. The traditional methods can generate EBs on a small scale (50 – 100 EBs per plate) and are not suited to large-scale requirements in therapeutic applications. Many methods have been developed to address these issues in the conventional methods of EB formation. Concave microwell arrays coated with non-adhesive polyethylene glycol, or polyurethane or polydimethylsiloxane (PDMS) have been developed to sustain in vitro EB cultures without media depletion12. Microwells can be used to regulate the EB size from 200 – 1000 μm. It has been reported that lineage-differentiation is specified by the size of EBs. Mesoderm and endoderm develop from larger EBs and ectoderm develops from smaller sizes. Cardiomyocyte differentiation seems to favor smaller EBs ranging from 100 – 500 μm in diameter. In another report, bioprinting technology was combined with hanging drops to give a reliable, robust and rapid method of making uniform EBs13. Bioprinting methods use both inkjet and laser-guided printing techniques to seed cells with minimal loss of viability and function. This offers a unique, reproducible and scalable alternative to generation of EBs. It gives a significantly lower variation and increased uniformity in EBs as compared to manual pipetting techniques of the stand-alone hanging drops methodology. Another alternative to large scale production of EBs is the stirred-suspension culture spinner flasks14. They facilitate the control of culture media, pH, and oxygen concentrations in the large scale culturing of EBs. This method enhanced the expression of cardiac specific genes, for example GATA binding protein 4 and a-cardiac myosin heavy chain. One or more of these various methods for controlled EB formation coupled with directed differentiation would prove instrumental in achieving significant results in regenerative medicine and in stem cell therapy.
We would like to thank Ms. Jacqueline Ng, Technical Sale Representative in Stemcell Singapore Pte Ltd. for providing us with free samples and sound technical support.
About the Authors
Dr. Suchitra Derebail is an experienced Scientist with over 11 years of experience in the fields of Stem Cell Biology, Virology and Molecular Biology. She has diverse research experience in several fields; lentiviral and baculoviral assays, stem cell reprogramming and differentiation assays. Her research interests are in the field of stem cell differentiation studies. Currently, she is working as Academic Staff at Republic Polytechnic in the School of Applied Sciences, where she facilitates several modules like Genetics and Cell Culture and is involved in training students through Final Year projects.
Ong Hong Boon, Ang Kailin, Nur Amilia Bte M Isa, Nur Ayuni Bte Hassan Jaya, and Orr Hui Min are final year students in the Diploma of Biomedical Sciences in Republic Polytechnic. They have worked as a team on this final year project, for a period of six months. They have also finished relevant modules on Genetics, Biochemistry, Molecular biology etc. Together with the experience garnered in this project, the team is well-equipped to handle stem cell culture work.
Casthri Krishnamurthy has worked as Technical Support Officer at Republic Polytechnic for the past five years. He was responsible for organizing and running several labs pertaining to cell cultures, cell cycle and oncology, stem cells and tissue engineering. He was instrumental in providing vital technical and logistical support for this project. Currently, he is working as a biomedical research engineer, specializing in the area of biosensing at Delta Electronics (Singapore) Pte Ltd.
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