Perspective Chapter: Two-Dimensional and Three-Dimensional Culture of Human Pluripotent Stem Cells

Abstract

Human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold immense potential for various biomedical research in both academic and clinical applications. This chapter provides a comprehensive review of culturing techniques for hPSCs, covering two-dimensional (2D) adherent culture, three-dimensional (3D) suspension culture, and the utilization of hydrogel scaffolds in 3D hPSC culture. Furthermore, it explores the application of advanced 3D cell manufacturing techniques to facilitate the production of large quantities of high-quality hPSCs, catering to the needs of advanced biomedical applications. By addressing these topics, this chapter aims to present a comprehensive overview of diverse cultivation methods and their wide-ranging applications in hPSC research, encompassing fundamental studies and advanced biomedical investigations.

1. Introduction

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) [1] and human induced pluripotent stem cells (hiPSCs) [2, 3]. These cells are characterized by unique features such as indefinite self-renewal and the remarkable capacity to differentiate into all the cell types in the body. Therefore, the hPSCs represent invaluable cell sources for fundamental stem cell research and a wide range of biomedical applications, including cell therapies, tissue engineering, drug discovery, and disease modeling [4, 5, 6, 7, 8]. In addition to hPSCs, tissue-derived adult stem cells can serve as valuable resources for stem cell therapies. These multipotent stem cells, including the stem cells isolated from fetal tissues, mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs), exhibit the capacity to differentiate into restricted cell types within their respective lineages [9]. The two-dimensional (2D) adherent culture of hPSCs is a commonly employed in research laboratories for fundamental stem cell studies. The 2D adherent cell culture methods can only produce the cells in limited quantities. However, all biomedical applications require significantly larger quantities of cells [10]. For example, the treatment with one patient of myocardial infarction typically necessitates approximately 10⁹ cardiomyocytes. Similarly, the treatment of one diabetic patient requires around 10⁹ β cells. In the case of blood transfusions, approximately 10¹² red blood cells are needed. Moreover, the engineering of a human-size liver and the screening of a library with one million compounds at once typically require approximately 10¹⁰ cells [11, 12]. Researchers have explored suspension culture methods [13, 14, 15, 16, 17, 18, 19] to scale up the production of hPSCs and their derivatives. Furthermore, alternative methods have been developed to enhance the scalability of hPSC culture, including cell microencapsulation within hydrogels such as alginate [20, 21, 22, 23] and thermoreversible hydrogels [24, 25]. These methods utilize a supportive matrix that closely mimics the natural extracellular environment, effectively preventing the formation of excessive cell agglomerations and reducing exposure to shear forces generated in the dynamic suspension culture. As a result, they exhibit high efficiency in expanding hPSCs [21, 24]. To facilitate the production of hPSCs on a large scale, implementing three-dimensional (3D) cell manufacturing processes is crucial. Extensive research has been dedicated to implementing 3D cell manufacturing processes for large-scale production [26, 27, 28, 29, 30].

2. Two-dimensional (2D) adherent and three-dimensional (3D) suspension culture of hPSCs

The hPSC culture media comprises vital nutrients, growth factors, and supplements that promote cell viability, proliferation, and maintenance of pluripotency. Initially, hPSC culture media contained serum and conditioned media [1, 31]. Currently, xeno-free and fully chemically defined media, such as mTeSR [32, 33] and Essential 8 (E8) [34], are commonly used for the cultivation and propagation of hPSCs. These media formulations eliminate the reliance on animal-derived components, offering a well-defined and controlled environment that supports the optimal growth and maintenance of hPSCs. For example, the E8 medium comprises eight essential components: DMEM/F12, insulin, FGF, L-ascorbic acid, selenium, TGFβ, transferrin, and NaHCO₃. DMEM/F12 serves as the base medium, providing a nutrient-rich foundation. Insulin and FGF2 play crucial roles in promoting the survival and proliferation of hPSCs. L-ascorbic acid acts as a stimulant for cell proliferation. Selenium is vital for the sustained expansion of hPSCs. TGFβ is incorporated to enhance pluripotent marker expression and ensure consistent long-term culture stability. When combined with a ROCK inhibitor (RI, Y27632) [35], transferrin improves initial survival and supports high cloning efficiency. NaHCO₃ is used for pH adjustment of the medium. These xeno-free and fully defined media efficiently support the expansion of hPSCs, enabling the transition of these cells from basic laboratory research to clinical applications.

The commercial hPSC cell lines, including hESCs and hiPSCs, can be purchased from WiCell Research Institute, the American Type Culture Collection (ATCC), or the European Collection of Authenticated Cell Cultures (ECACC). The equipment and supplies for hPSC maintenance include cell culture incubator, biosafety hood, 4°C refrigerator, −20°C freezer, −80°C freezer, liquid nitrogen dewar, inverted microscope, water bath, centrifuge, cell-freezing container, cryovials, pipette set with various tips, and different sizes of tubes (e.g., 1.5, 15, and 50 mL) along with standard cell culture plates (e.g., 6-well cell culture plates or 24-well cell culture plates) and low-attachment cell culture plates (e.g., low-attachment 6-well cell culture plates or low-attachment V bottom or U bottom 96-well cell culture plates). Additionally, the following materials and reagents are required to culture hPSCs: Essential 8™ medium (Gibco, or STEMCELL Technologies), Matrigel matrix (Thermo Fisher Scientific, Corning Life Sciences, or VWR), Rock inhibitor (Tocris Bioscience, Selleck Chemicals, or MilliporeSigma), Accutase (STEMCELL Technologies, MilliporeSigma, or Invitrogen), EDTA (0.5 M), pH 8.0, RNase-free (Thermo Fisher Scientific, VWR, or Cayman Chemical), DPBS (no calcium, no magnesium, Gibco, MilliporeSigma, or VWR), Dimethyl sulfoxide (Sigma-Aldrich, Thermo Fisher Scientific, or VWR), Penicillin-streptomycin solution (Corning Life Sciences, MilliporeSigma, or VWR), and 70% ethanol.

2.1 2D cell culture methods

The 2D adherent culture of hPSCs in cell culture plates or Petri dishes is a commonly employed method in laboratories for fundamental stem cell studies such as cell expansion and differentiation (Figure 1). The culture substrate plays a significant role in supporting hPSC attachment and proliferation, maintaining pluripotency. hPSCs were initially cultured on a layer of feeder cells, which refer to mitotically inactivated mouse embryonic fibroblast (MEF) or human fibroblast feeders that support hPSC growth and maintenance [1, 37]. Due to the significant variability and irreproducibility associated with the feeder-dependent methods, a feeder-independent culture protocol was developed [33], in which the hPSCs were cultured on extracellular matrix (ECM) rather than the feeder cells. Matrigel, an Engelbreth–Holm–Swarm mouse sarcoma tissue extract, has emerged as one of the most extensively utilized extracellular components for feeder-free culture of hPSCs in laboratories [5, 31, 33, 36]. Matrigel is a partially chemically defined and xenogeneic substrate encompassing collagens, laminin, and various other chemical compounds [38]. Therefore, xenogenic-free, chemically defined ECM, such as laminin [39, 40], vitronectin [41], and synthetic surfaces [42], was developed to facilitate hPSC expansion and differentiation on the 2D adherent substrates. hPSCs cultured in 2D systems provide valuable insights into their molecular characteristics and differentiation potentials.

Figure 1.

2D cell culture of human pluripotent stem cells [33, 36]. (a–c) the 2D cell culture is prepared for passaging. The colony and cell morphology when observed through 2× (a), 4× (b), and 10× (c) objectives, respectively. The center of the colonies appears denser and brighter and cells at the center of the colony may appear smaller. Scale bar: 100 μm [33]. (d-f) Following plating, the cells quickly adhere to the plate within a few minutes (d), spread within 2 hours (e), and subsequently grow as colonies within 24 hours (f). Scale bars: 50 μm [36].

The 2D adherent cultures are widely used in laboratories for hPSC expansion and differentiation. The procedures involve several steps to ensure successful cell seeding, cell growth, and maintenance. These steps include (1) thawing frozen cells, (2) seeding cells on ECM-coated culture plates, (3) cell maintenance, (4) cell passaging, and (5) cell freezing for storage. Throughout these processes, it is important to exercise caution to minimize the risk of cell contamination and stress. Adhering to aseptic techniques, maintaining proper culture conditions, and strictly following established protocols will help to preserve the quality of hPSCs, especially for maintaining their pluripotency for long-term culture. Detailed protocols for culturing hPSCs on a 2D surface are provided below, offering step-by-step guidance and best practices.

E8 medium preparation: The E8 basal medium should be stored in a refrigerator at 4°C. Aliquot the E8 supplements into 1-mL tubes and store them in a freezer at − 20°C or − 80°C. To prepare the E8 full medium, mix 48.5 mL of the E8 basal medium with 1 mL of the E8 supplement and 0.5 mL of the penicillin-streptomycin solution.

Matrigel preparation: Aliquot the Matrigel into small volumes (e.g., 200 or 500 μL) and store them in a − 80°C freezer for a long time. The Matrigel was left in a 4°C refrigerator overnight to dissolve, resulting in a viscous liquid. To aliquot the Matrigel, place the Matrigel and all other required tubes in an ice bag in the biosafety hood in case the Matrigel forms the hydrogel during the procedures. To prepare the Matrigel coating medium, dilute the Matrigel with cold DMEM/F12 basal medium to a final concentration of 100 μg/mL. To coat the cell culture plate, add 1 mL of the Matrigel coating solution into one well of the 6-well plate. Place the cell culture plate at room temperature in the biosafety hood for at least 1 hour or place it in the incubator at 37°C for at least 30 minutes.

2.1.1 Thawing frozen cells and seeding on the 2D surface

1. Retrieve one cryovial of hPSCs from the liquid nitrogen dewar. Note: Ensure you have received specific training and wear appropriate personal protective equipment (PPE) before handling liquid nitrogen.

2. Submerge the bottom of the cryovial containing the cells into a 37°C water bath immediately. Gently shake the cells for about 3–5 minutes. Monitor the cells closely, and once the ice has dissolved into small pieces, carefully remove the cryovial from the water bath. Disinfect the exterior of the cryovial by gently pouring 70% ethanol.

3. Place the cells inside a biosafety hood. Utilizing a 1-mL pipette, carefully transfer the cells into a 15-mL tube. Following this, add 9 mL of E8 full medium supplied with 10 μM RI. Gently mix the tube to ensure proper distribution of the cells and medium.

4. Centrifuge the cell suspension at 300 g for 5 minutes.

5. Carefully discard the supernatant, ensuring complete removal. Next, add 2 mL of E8 full medium supplied with 10 μM RI. Resuspend the cells by gently pipetting a couple of times.

6. Remove the Matrigel coating medium from the cell culture plate. Note: The Matrigel coating medium can be recycled and used within 2 weeks.

7. Transfer the 2 mL of cells into one Matrigel-coated well of the 6-well plate. Culture the cells at 37°C and 5% CO₂ in the incubator. Note: To ensure even distribution of the cells, gently shake the cell culture plate back and forth a couple of times.

8. On the second day, replace the medium with 2 mL of fresh E8 full medium. Continue culturing the cells for an additional 3–4 days. Observe the cell morphologies using an inverted microscope and allow the cells to reach approximately 80% confluency before the following passage. Remember to change the medium daily throughout this period.

2.1.2 2D cell passaging (passaging cell clusters using EDTA solution)

1. Before passage, add RI to each well at a final concentration of 10 μM. Then, put the cells back in an incubator for culture for at least 1 hour. Note: RI treatment can significantly improve cell viability after passage.

2. Remove the cell culture medium and add 1 mL of DPBS (without Ca²⁺ or Mg²⁺) to each well. Gently shake the plate to ensure proper distribution, then carefully remove the DPBS.

3. Add 1 mL of 0.5 mM DETA solution to each well. Allow the cells to remain undisturbed at room temperature for approximately 5–7 minutes.

4. Remove the EDTA solution from each well. Using a 1-mL pipette, gently pipette the cells up and down with 1 mL of E8 full medium supplemented with 10 μM RI.

5. Transfer 200 μL cell solution to a new Matrigel-coated well and add 2 mL fresh E8 full medium supplemented with 10 μM RI.

6. Place the cells in the incubator. To ensure even distribution of the cell clusters within the wells, gently shake the cell culture plate back and forth several times.

7. On the second day, replace the medium with 2 mL of E8 full medium. Continue culturing the cells for an additional 3–4 days. Observe the cell morphologies using an inverted microscope and allow the cells to reach approximately 80% confluency before the following passage. Change the medium daily.

2.1.3 Cell freezing (when ordering new cells, it is necessary to expand them through 2–3 passages and then freeze the cells for banking)

1. Prepare the cell freeze medium by combining 10% DMSO with 90% E8 full medium supplemented with 10 μM RI. Note: Add the DMSO into the E8 medium slowly, drop by drop.

2. The EDTA-based procedure described above was used to dissociate the cells into cell clusters. Note: Freezing cell clusters instead of single cells can enhance the efficiency of cell recovery.

3. Collect the cell clusters into one 15-mL tube. Centrifuge the cells at 300 g for 5 minutes.

4. Remove the supernatant from the tube.

5. Add 1 mL of cell freeze medium to the tube. Next, resuspend the cell clusters by gently pipette a couple of times. Finally, transfer the resulting cell solution into a cryogenic vial.

6. Place the cryogenic vial into the cell-freezing container.

7. Transfer the cell freezing container to a − 80°C freezer and leave it overnight.

8. On the second day, transfer the frozen cells to a liquid nitrogen dewar for long-term storage.

In summary, 2D cell culture methods are widely used in laboratories for basic stem cell research. For hPSC maintenance, it is essential to exercise caution to minimize the risk of cell contamination and cell stress. Preserving the high quality and pluripotency of hPSCs requires strict adherence to aseptic techniques, meticulous maintenance of culture conditions, and rigorous adherence to established protocols. Despite their extensive use, 2D culture approaches have inherent limitations. These systems fail to fully replicate the intricate 3D microenvironment in vivo, which hampers accurate modeling of cell-cell interactions, morphogenesis, and organ development. As a result, researchers have developed 3D suspension culture methods to address these questions.

2.2 3D suspension culture methods

In laboratories, 3D suspension culture methods have also emerged as valuable tools for studying hPSCs’ expansion and differentiation in a more physiologically relevant environment (Figure 2). This approach allows cells to interact with neighboring cells and the extracellular matrix, promoting cell-cell interaction and tissue-like organization. The 3D culture enables the formation of more complex structures, such as embryoid bodies (EBs) or organoids, which are self-organized 3D tissues with multiple cell types. Organoids derived from hPSCs have demonstrated remarkable cellular organization and functionality, offering great potential for cell therapies, disease modeling, and drug screening. The 3D suspension culture is widely used for hPSC and hPSC-derived organoid cultures [8, 43, 44, 45, 46, 47, 48]. In this system, the cells can be cultured in low-attachment cell culture plates or spinner flasks. The detailed protocols for culturing hPSCs on 3D suspension are provided below, offering step-by-step guidance and best practices.

Figure 2.

3D suspension culture of human pluripotent stem cells [19]. (a–e) Single cells were seeded in the 3D suspension culture on day 0 (a); the formation and growth of cell spheroids from day 1 to day 4 (b–e). (f) Undesirable cell spheroid agglomeration formed in the center of the dish. Scale bars: 500 μm.

2.2.1 2D to 3D suspension passage cells

1. Before passage, add RI to each well of the cells at a final concentration of 10 μM. Subsequently, return the cells to an incubator at 37°C and allow them to incubate for at least 1 hour.

2. Discard the cell culture medium from each well.

3. Add 1 mL of DPBS to each well, gently shaking the plate, and then remove the DPBS from each well. Note: It is necessary to perform a single wash of the cells with DPBS. The resident cell culture medium will neutralize the Accutase activity.

4. Add 1 mL of Accutase solution to each well. Place the cells at 37°C in the incubator for 3–5 minutes.

5. Observe the cell statue under the miscopy. When you see many cells detached from the bottom of the plate, add 2 mL of E8 full medium supplemented with 10 μM RI. Gently pipette the cells into single cells using a 1-mL pipette.

6. Perform cell counting by combining 10 μL of the cell solution with 10 μL of trypan blue. Transfer 10 μL of this mixture to a cell counting plate. Utilize the automated cell counter to measure cell viability and concentration.

7. Centrifuge the single-cell solution at 300 g for 5 minutes.

8. Carefully discard the supernatant from the tube. Add 1 mL of E8 full medium supplemented with 10 μM RI. Resuspend the cells by pipetting a couple of times.

9. Calculate the cell number needed for the following experiments.

2.2.2 For 3D suspension culture in low-attachment 96-well plate (many organoid culture protocols begin by generating hPSC spheroids using a low-attachment V bottom or U bottom 96-well plate)

1. Calculate the cell concentration and dilute the cells in E8 full medium supplemented with RI. Seed around 3000–6000 cells per 200 μl medium per well in the low-attachment 96-well plate.

2. Put the cells at 37°C in an incubator for culture. The cells will go down to the bottom of the well and form one cell cluster within 1 hour.

3. On the second day, the cells should form a uniform spheroid in each well. Carefully remove 150 μL medium from each well and add 150 μL E8 full medium.

4. Change the medium daily and process to organoid differentiation on day 3–5 by replacing the medium with an organoid-specific differentiation medium.

2.2.3 For 3D suspension culture in low-attachment 6-well plate

1. Transfer 1×10⁵ cells into a single well of the low-attachment 6-well plate. Add 2 mL of fresh E8 full medium supplemented with 10 μM RI.

2. Place the cell plate onto a shaker inside the incubator for suspension culture. The cells will form small cell spheroids within 24 hours.

3. Passage the cells on days 5–7. The final concentration of the cells in the 3D suspension culture is about 1–2 million cells per mL.

2.2.4 3D to 3D suspension passage cells

1. Add RI to the cells at a final concentration of 10 μM. Subsequently, return the cells to the incubator and culture them for at least 1 hour.

2. Collect the cells and transfer them to 15-mL tubes.

3. Centrifuge the cells at 200 g for 3 minutes.

4. Remove the supernatant, wash the cells once using DPBS, and add 5–10 Accutase solution to digest the cell spheroids.

5. Put the sample at 37°C for about 10–15 minutes and dissociate them into single cells using a 1-mL pipette by carefully pipetting up and down several times.

6. Add equal E8 full medium supplemented with RI to neutralize the Accutase activity.

7. Perform the cell counter and seed the cells at a concentration of 1×10⁵ cells.

Biomedical applications require large quantities of cells of high quality. To scale up the production of hPSCs, researchers have explored suspension culture methods using trimethyl ammonium-coated polystyrene microcarriers [14] or Matrigel-coated microcarriers [13]. However, it has been observed that the cell expansion rate tends to decrease gradually over long-term culture on microcarriers, and there can be challenges associated with detaching the cells from these microcarriers [13, 14]. To address these challenges, researchers have developed 3D suspension culture techniques for hPSCs without using microcarriers [15, 16, 17, 18, 19]. hPSCs have been successfully cultured as floating aggregates in static conditions using low-attachment cell culture plates or in suspension culture with regular agitation of the culture vessel, such as spinner flasks. Currently, the 3D suspension culture of hPSCs and hPSC-derived organoids in spinner flasks is also commonly applied in laboratories.

2.3 3D culture with hydrogel scaffolds

Hydrogel scaffolds like alginate [20, 21, 22, 23], or thermoreversible hydrogels [24, 25] have been developed to culture hPSCs in a more physiologically relevant microenvironment. These hydrogel scaffolds can be used as physical barriers to isolate cells from agglomeration and hydrodynamic stresses generated from the dynamic suspension. These methods enhance the scalability of hPSC culture and exhibit high efficiency in expanding hPSCs [21, 24]. For example, a thermoreversible hydrogel-based cell culture system has been developed to expand and differentiate multiple cell lines of hPSCs [24, 25]. In this system, single cells were seeded within this thermoreversible PNIPAAm-PEG hydrogel matrix (Figure 3). As the hydrogel is very soft, the single cells can clonally grow into uniform spheroids within 3–5 days by deforming the scaffolds to create spaces. The hydrogel eliminating the cell agglomeration and hydrodynamic stresses could significantly improve the culture efficiency. The hydrogel scaffold enabled serial expansion of hPSCs with good cell viability, growth rate, yield, and purity. Moreover, after expansion, the hydrogel can be easily dissolved into liquid by adding cold DPBS to the well. Then, the cell spheroids can be easily harvested and centrifuged for the following passage procedures. This method provides an efficient, defined, scalable, and good manufacturing practice-compatible approach for hPSC production and differentiation [24].

Figure 3.

3D culture of hPSCs in thermoreversible PNIPAAm-PEG hydrogels [24]. Phase images depict cell morphologies following culture in PNIPAAm-PEG hydrogel. Single-cell seeding densities were 2.5×10⁵, 1.0×10⁶, or 2.5×10⁶ cells per mL, respectively, and cultures were maintained for 4 days in either mTeSR or E8 medium with 1-day or 4-day RI. Scale bar: 250 μm.

Additionally, alginate hydrogels offer tunable mechanical properties and can encapsulate hPSCs to form 3D structures. Alginate hydrogel tube cell culture system (AlgTubes) [21, 22, 23] provides cells with a culture microenvironment that aligns with their physiological needs. In this system, hPSCs are seeded in microscale alginate hydrogel tubes (Figure 4). Within 24 hours, these hPSCs can form small cell clusters, enhancing cell viability post-seeding. The diameter of the tubes can be adjusted to approximately 200–400 μm, allowing nutrients and metabolic waste to diffuse freely through the hydrogel tube walls. The design of AlgTubes represents a paradigm shift in cell culture efficiency, as evidenced by improvements in various aspects such as growth rate, viability, yield, and genetic and phenotype stability. Furthermore, AlgTubes can be easily adapted for scalable expansion in bioreactors, showcasing the high potential for producing hPSCs and their derivatives with exceptional quality and quantity, making them ideal for various biomedical applications.

Figure 4.

3D culture of hPSCs in Algtubes [21]. Phase images illustrate the cell morphologies during culture in Algtubes. Cells were seeded at densities of 1×10⁶, 2×10⁶, 5×10⁶, or 10×10⁶ cells per mL and were cultured for 9 days. Within 24 hours, individual cells aggregated to form small clusters. These clusters subsequently grew into spheroids by day 5, which further evolved into fibrous cell masses by day 9.

2.4 3D cell manufacturing for advanced biomedical applications

HPSCs hold great promise for a range of cutting-edge biomedical applications, all of which necessitate substantial quantities of high-quality cells [11, 12]. (1) hPSCs can differentiate into diverse cell types, such as cardiomyocytes and neurons, with applications in regenerative medicine and organ transplantation. For instance, treatment of myocardial infarction in a single patient often demands roughly 10⁹ cardiomyocytes. (2) hPSCs can be derived from individual patients, paving the way for personalized disease modeling and high-throughput drug screening. Notably, screening a library containing one million compounds at once typically demands around 10¹⁰ cells. (3) hPSCs offer the potential to produce substantial quantities of blood cells, including platelets. For example, approximately 10¹² red blood cells are required for blood transfusions.

Advancing the mass production of hPSC-based therapeutics toward a scalable and cost-efficient process will facilitate widespread adoption in all biomedical applications. The field of 3D cell manufacturing has opened exciting possibilities for large-scale production of hPSCs and their derivatives. Developing the 3D cell manufacturing processes requires incorporating approaches that allow for the precise control of culture conditions, such as nutrient and oxygen supply, pH, and mechanical forces, as well as precise monitor the cell conditions, including cell viability, cell states, and cell function [26, 27, 28, 29]. Extensive research has been dedicated to implementing 3D cell manufacturing processes using bioreactors for large-scale production of hPSCs [27, 30, 49, 50]. For instance, bioreactors such as the Cellspin Integra Biosciences spinner flask [51], the NDS Technologies Bioreactor [52], and the Able Bioreactor [53] have successfully generated clinically relevant cell numbers. While the agitation of culture in bioreactors can improve mass transport and reduce cell agglomeration, it can also introduce hydrodynamic stresses, such as shear stresses, which may negatively impact hPSC expansion and differentiation. The variations in the stirred-tank bioreactor are influenced by numerous factors, including the bioreactor design, such as impeller geometry and size, as well as vessel geometry and size. Therefore, there is a critical need for automated 3D cell manufacturing in advanced biomedical applications, enabling the large-scale production of hPSCs in accordance with current good manufacturing practices (cGMP) and strict safety assessment criteria. In summary, advancements in this field continuously broaden our comprehension of hPSCs and their wide-ranging potential in addressing diverse human diseases and industrial demands.

3. Conclusion

This chapter has provided a comprehensive overview of the different culturing methods used in stem cell research and their biomedical applications. The 2D cell culture methods have been extensively employed in laboratories for fundamental stem cell research. Detail protocols for hPSC culture in 2D adherent substrates were summarized. Furthermore, 3D suspension culture methods have demonstrated their value in basic stem cell research and advanced biomedical studies. The utilization of hydrogel scaffolds, including alginate and thermoreversible hydrogels, has enabled the development of 3D culture systems for hPSCs expansion and differentiation. Additionally, the application of 3D cell manufacturing techniques has shown promise in producing large quantities of high-quality hPSCs for advanced biomedical applications. This chapter has offered readers a comprehensive understanding of the diverse culturing methods and their roles in stem cell research, ranging from fundamental studies to cutting-edge biomedical advancements. This knowledge serves as a foundation for readers interested in delving deeper into stem cell biology and biomedical applications, empowering them to make valuable contributions to the field.

4. Troubleshooting

Problem 1: Matrigel forms hydrogel during handling.

Solution 1: Thaw the frozen Matrigel at 4°C overnight. Ensure all handling steps, including aliquoting and preparing the Matrigel coating solution, are performed while keeping the Matrigel on ice.

Problem 2: In 2D culture, cell colonies cannot be detached from the plate.

Solution 2: Appropriately extend EDTA treatment time.

Problem 3: In 2D culture, there is a low cell survival rate after cell plating.

Solution 3: Ensure RI is added before cell passage and continue including RI in the cell culture medium within 24 hours after passage.

Problem 4: In 3D suspension culture, lots of large cell aggregations form.

Solution 4: Appropriately increase agitation rate.

Appendices and nomenclature

hPSCs	human pluripotent stem cells
hESCs	human embryonic stem cells
hiPSCs	human-induced pluripotent stem cells
MSCs	mesenchymal stem cells
HSCs	hematopoietic stem cells
2D	two-dimensional
3D	three-dimensional
E8	essential 8
RI	ROCK inhibitor
MEF	mouse embryonic fibroblast
ECM	extracellular matrix
DMSO	dimethyl sulfoxide
PPE	personal protective equipment
EBs	embryoid bodies
AlgTubes	alginate hydrogel tube cell culture system
cGMP	current good manufacturing practices