Is It Time to Start Transitioning From 2D to 3D Cell Culture?

1Two dimensional (2D) cell culture has been the method used to culture cells since the early 1900s, which plays a vital role in research but has many limitations due to 2D models inaccurately representing tissue cells in vitro. Another method known as 3D cell culture has shown improvements in studies targeted toward morphology, cell number monitoring, proliferation, response to stimuli, differentiation, drug metabolism, and protein synthesis. All of this is made possible by 3D cultures’ capability to model a cell in vivo while being cultured in vitro. 3D cell culture has many applications such as cancer research, stem cell research, drug discovery, and research pertaining to other types of diseases, which is more popular today than ever (Figure 1). Table 1 compares the different aspects of 2D and 3D cell culture and explains the advantages and disadvantages of both methods. Furthermore, 3D culture offers several methods of cell culture depending on the type of experiment being performed.

2Scaffold based techniques such as hydrogel-based support, polymeric hard material-based support, hydrophilic glass fiber, and organoids provide an array of advantages. Hydrogels are unique because of their ability to mimic the ECM while allowing soluble factors such as cytokines and growth factors to travel through the tissue-like gel. Hydrogels are also versatile since they can be used to create spheroids and can be prepared in multiple ways depending on the experiment being performed. Both natural and synthetic hydrogels exist, with natural gels commonly being made with natural polymers such as fibrinogen, hyaluronic acid, collagen, Matrigel, gelatin, chitosan, and aginate. Natural gels made of collagen have been used to model 3D tumors via MCTS where the cells were embedded in the gel. The study concluded that the 3D model allowed for drug screening as well as noticed differences in cell shape, density, and drug sensitivity when compared to cells cultured on the traditional monolayer. Synthetic hydrogels are typically made with synthetic polymers made from polyethylene glycol (PEG), polylactic acid (PLA), or poly(vinyl acetate) (PVA). Polymeric hard scaffolds are an important tool in studying cell-to-ECM interactions due to the scaffold’s ability to replicate the structure of the ECM. A study showed that HepG2 liver cells cultured using a 3D polymeric hard scaffold were less affected by cytotoxic compounds and had greater viability than those grown in 2D. Furthermore, polymeric hard scaffolds are extremely useful in studying tissue regeneration as well as testing tumor cell treatments. Hydrophilic glass fibers are important for modeling 3D tumors testing antibodies, invasion, as well as tracking cell migration. SeedEZ discovered by Lena Biosciences is such an inert and transparent glass microfiber scaffold (Figure 2), which allows for various cell types to be seeded at once in order to create different 3D layers within the cell. Compared with other 3D cell culture systems, such as 3D Matrigel culture drops, SeedEZ promotes cell-cell interaction and formation of 3D cell network more efficiently. By taking these advantages, SeedEZ represents the most effective tools for cancer research and drug testing. The use of hydrophilic glass fibers are still to be further explored, but offer an abundance of potential. Organoids aggregate into spheroids by forming ECM fibers that link single cells together via integrin binding and mimic the microenvironment of certain organs to allow researchers to model human diseases through the use of patient-derived pluripotent stem cells. Furthermore, researchers are able to grow tumor models using organoids through the use of patient derived tissue cancer cells. This allows scientists to model the patient’s tumor in order to test treatments on a patient-to-patient basis. Lastly, organoids have shown signs that one day they may be able to aid in an alternative organ transplantation method. Organoids are changing the way in which researchers study human development, as well as test new disease treatments.

3Scaffold-free techniques including hanging drop microplates, magnetic levitation, and spheroid microplates with ultra-low attachment coating are unique in their ability to freely grow with no scaffold and provide special advantages as a result. Hanging drop plates allow the formation of spheroids via self-aggregation through the use of gravity. The spheroids hang in open bottomless wells which are often enclosed in the bottom of the plate in order to regulate the environmental humidity of the cells. Hanging drop plate methods have a wide range of uses due to their replicability. A study was conducted in which cardiac spheroids were created by co-culturing endothelial cells, fibroblasts, and cardiomyocytes derived from induced pluripotent stem cells. The results showed a cell culture model in which toxic effects in human heart tissue could be studied due to how closely the cardiac spheroids resembled in vivo features of the human heart. Magnetic levitation is performed by injecting cells with magnetic nanoparticles allowing cells aggregate into a spheroid when exposed to an external magnet. This creates a concentrated cell environment in which ECM can be synthesized, and analyzation via western blotting and other biochemical assays can be performed. Furthermore, the external magnet can be used manipulate the 3D culture, allowing for special control and more advanced environments. Overall, magnetic levitation allows both basic and advanced environments to be replicated, thus making it a very versatile technique. Spheroid microplates with ultra-low attachment coating are commonly used to study tumor cells as well as grow multicellular cultures due to the large volume. Studies show that multicellular spheres that were grown from two NSCLC cells display very different growth characteristics when compared to 2D cell cultures. The cells exhibited multidrug resistance, displayed stem-cell like traits, and cell motility was increased. Furthermore, tumor cells derived from breast cancer cells display in vivo characteristics that are useful when testing treatments.

4A common tool used in research is the use of animal models. Mouse models are commonly used in research to test new drugs and treatment strategies especially in cancer research. 3D culturing techniques have allowed researchers to model tumors and organs in order to perform drug treatment tests on them. Experts suggest that as these models continue to improve and become more commonplace, less animal models will need to be used.

53D cell culturing methods are beginning to outperform old 2D cell culture methods despite the fact that 3D culture is still in its infancy stages. Furthermore, each 3D culturing method comes with a unique set of advantages that can be implemented depending on the desired experiment. Table 2 displays a comparison between hydrogel-based support, polymeric hard material based support, hydrophilic glass fibers, magnetic levitation, and spheroids with ultra-low attachment coatings.

6Drug discovery is the most important aspect in the fields of medicine and pharmacology, but often takes an extensive amount of time as well as money and yields low success rates when testing new medicines in animal models and in preclinical trials. Due to low success rates, less than half of all drugs in Phase II and Phase III clinical trials are successful indicating the desperate need for new methods and technologies that improve the efficacy of drug discovery. Animal models tend to be expensive, whereas assays using cultured cells have proven to be to be easily replicable, quick, and cost-effective. The most commonly used method in drug discovery to date is the use of 2D cell cultures. 2D cell cultures have aided in the discovery of many biological and disease processes but are unable to mimic the complicated microenvironment cells experience in tissue. Drug discovery relies on understanding the link between cells and the ECM in which they interact. ECM molecules include matrix proteins, glycoproteins, glycosaminoglycans, proteoglycans, ECM-sequestered growth factors, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) as well as other secreted proteins. These growth factors and proteins play key roles in regulating cell proliferation, migration, differentiation, adhesion, and survival. Furthermore, the structure of the ECM can affect the cell’s response to drugs by changing a drug’s mechanism of action, amplifying drug efficacy, or by boosting the cells affinity for drug resistance.

7In order to predict the effectiveness of a drug on a cell, a 3D culture model would have to mimic the microenvironment of tissue in which cells can proliferate, aggregate, and differentiate. Cells cultured in 3D have displayed different responses to drugs than cells cultured in 2D for several reasons. Differences in physical and physiological properties between 2D and 3D cultures cause 2D cells to be more susceptible to the effects of drugs than 3D cells due to the fact that 2D cells are unable to maintain a normal morphology like 3D cells can. Another reason 2D cells are more sensitive to drugs than 3D cells is because of the difference in the organization of surface receptors on the cell. Drugs often target certain receptors on the surface of cells. Differences in structure and spacial arrangement of surface receptors likely effect the binding efficacy of drugs to the receptors eliciting different responses. Third, cells cultured in 2D are often all at the same cell stage whereas 3D cells are often in different cell stages much like cells in vivo. In 3D cells, difference in cell stage likely means that there are proliferating cells available in the outer region in the cell. Many drugs require cell proliferation to be effective, favoring 3D cell culture. Lastly, the difference in shape between 2D and 3D cells causes a difference in local pH levels within the cells as a result of 3D cells having greater depth than 2D cells. It has been proven that lower intracellular pH levels cause a reduction in drug efficacy, contributing to drug resistance.

8Metabolic profiling is used to demonstrate metabolic cooperation between varying cell types and is becoming a popular technique in 3D culture models due to the accuracy of the results when compared to cells in vivo. Previously, 2D culture models have been used to test cancer metabolism but recent studies suggest 3D culture models provide more insight when testing the efficacy of new drugs. Through profiling, researchers have discovered that drug treatments sometimes kill all the cells in 2D culture monolayer but only kill some of cells that make-up the protective layer of spheroids in 3D models. The extra dimension in 3D culture has helped researchers understand the flaws present in 2D models that cause lower rates of drug efficacy relative to in vivo trials. Furthermore, researchers have concluded that metabolic profiling in 3D culture is inherently different than 2D culture due to a reduced sensitivity to ATP synthase (a common metabolic inhibitor). Researchers concluded that this difference would cause a distinction in metabolic profiles due to differences between the responses to various chemotherapeutics in 2D and 3D.

93D cell culture has become one of the top methods of choice in drug discovery due to the fact that 3D cell cultures allow cell-to-cell and cell-to-matrix interaction much like the interactions cells in vivo experience. Furthermore, 3D culture models using human cells avoids the use of mouse models which are often expensive and inaccurately depict the effectiveness and side effects of drugs.

10Stem cells are commonly used as a means of regenerative medicine and cell therapy applications. In clinical applications however, 2D cell culture techniques have proven to be ineffective when using stem cells. This is a result of the 2D culture’s inability to accurately replicate the in vivo microenvironment of stem cells. Furthermore, MSCs often decrease in replicative ability as time progresses when culturing in 2D, invalidating any chance of them being used in large scale randomized clinical trials despite them showing beneficial effects in small-scale studies. When cultured in spheroids however, MSCs display a different morphology than 2D cultured MSCs. MSCs cultured in spheroids have gene expression patterns unlike those cultured in 2D. They display the upregulation of multiple genes associated with stress response, inflammation, redox signaling, hypoxia, and angiogenesis.

11Through the use of spheroid cultures, MSC-based therapeutics have greatly improved. In relation to 2D cultures, MSC spheroid cultures showed improvements such as an increase in the paracrine secretion of cytokines, more robust antiapoptotic and antioxidative capacities, and rising levels of ECM proteins. In addition, anti-inflammatory, tissue regenerative and reparative effects, and higher rates of posttransplant survival of MSCs have been observed as a result of MSC spheroid cultures. To further test the effectiveness of MSC spheroids, MSC spheroids were injected into the kidneys of mole rats with ischemia reperfusion-induced acute kidney injury where the results were recorded. The results post-injection showed that compared to 2D cultured cells, MSC spheroids were more effective in guarding the kidney against apoptosis, lessening tissue damage, bolstering vascularization, and alleviating renal function compared with 2D cultured cells.

12It has been recently discovered that pluripotent stem cells can be used to grow organoids that could potentially be used as a source of analogous tissue for transplantation in humans someday. Researchers have successfully demonstrated that renal organoids derived from pluripotent stem cells can be transplanted under the renal capsules of adult mice. The organoid resembled the structures of a kidney in vivo and upon transplantation, the glomeruli were vascularized quickly showing promising signs toward an alternative kidney replacement strategy. Using organoids derived from pluripotent stem cells, the future for alternative organ transplants in other organs is still being researched but remains optimistic.

13Organoids play an increasingly important role in the study of genetic diseases due to their ability to model different regions of the body. For example, a rectal organoid was used to model cystic fibrosis to study the effects of the transmembrane conductance regulator-modulating compounds and another set of tubular organoids was used to model kidney disease where it was found that the microenvironment played a key role in the cyst formation. Furthermore, organoids have proven to be useful models when studying neurodegenerative diseases such as Alzheimer and Parkinson disease. Brain organoids generated from pluripotent stem cells taken from Alzheimer patients when treated with β- and γ-secretase inhibitors, showed promising therapeutic effects.

14Lee Rubin, Ph.D. from the University of Harvard has begun to mass produce brain spheroids, as well as derive spheroids from patient cells to create a biobank in which treatments can be tested on a patient-to-patient basis. By growing an unlimited supply of tissue, rare diseases can be tested on endlessly in an effort to find cures. Paola Arlotta, Ph.D. is another researcher from Harvard who allows organoids to grow for long periods of time so that they can develop into multi-thousand brain cell organoids that contain several brain cell types. This has allowed her to extensively study brain cell interactions with each other, to help understand how the cells communicate. Furthermore, researchers have found a link between autism and irregularities in the regulation of genes that play a role in proliferation using organoids derived from patients with autism. Although no cure has been found for autism, 3D cell culture techniques such as organoids have allowed researchers to take the first step in the direction of improvement and will continue to help uncover the mysteries behind many of the diseases people face.

15Real time visualization and analysis can play an important role in many different types of experiments. An experiment was conducted in which a 3D microfluidic system was created to mimic the microenvironment of a cell during angiogenesis via the use of a hydrogel scaffold. The hydrogel was microinjected into the microfluidic system allowing for control over surface shear stress, the flow of interstitial fluid through the matrix, the effects of the cell culture scaffold, gradients involving non-reactive solutes, as well as allowed cells to be monitored in real time. Three different extracellular capillary morphogenesis assays were performed, and time-lapse videos were taken of the cells in real-time to provide evidence of the multifunctionality of the 3D microfluidic device. The implementation of the hydrogel allowed the cells to be cultured within the microfluidic device giving the user more control over the microenvironment.

16Perfusion systems have become a common way to replicate and monitor in vivo environments. In one investigation, a perfusion 3D cell culture microfluidic chip was created to monitor, and record real-time impedimetric biosensor changes as a result of cellular responses in oral cancer cells. Using a 3D agarose scaffold, the cells were encapsulated and cultured in a small chamber under perfusion of culture medium. The microenvironment was effective for studying cell proliferation and chemosensitivity of anti-cancer drugs in a non-invasive and real-time manner.

17Recent advancements of microfluidic chips have led to what is known as the organ-on-a-chip model (also known as organ chips). These organ chips overcome many difficulties currently presented in spheroids and organoids grown in ECM gels. Although spheroids and organoids are useful ways to model many types of cancers, they present limitations due to the lack of tissue-tissue interfaces and organ-level structures. Organ chips are created using computer microchip fabrication and are populated with living cells that resemble in vivo organ-level physiology and pathophysiology. This is made possible by constructing tissue-level and organ-level structures in vitro that function like tissues and organs do in vivo. Not only do organ chip models allow for better organ models, but they also permit high-resolution and real-time imaging making it easier to analyze in vitro biochemical, genetic, and metabolic activities present in human tissue. Some human organs that have been successfully modeled on organ chip devices include: kidney tubules, small intestine, bronchioles, liver, BBB, lung alveoli, and bone marrow. Not only can these organs be modeled, but more importantly they can give accurate organ-level responses to many stimuli including drugs, toxins (66), radiation, cigarette smoke, and pathogens. Furthermore, therapeutic strategies and drug development are being tested on organ-chip models that mimic organs with diseases such as thrombosis, inflammatory bowel disease, asthma, and barth syndrome.

18A highly detailed experiment was conducted that constructed a blood-brain barrier chip (BBBC) model that mimicked the in vivo structure of micro blood vessels in the brain through the use of a type 1 collagen hydrogel. Endothelial cells, pericytes, and astrocytes from neonatal rats were cocultured in the collagen matrix to study cell interactions in the brain microvasculature as well as test new drugs for neurovascular diseases. The BBBC fluid flow used gravity and resistance in a paper-based resistor as a driving force rather than a pump. The fluid flow made the BBB more accurate than previous static 2D models since the media flow provided mechanical cues and facilitated mass transfer allowing functional maintenance of the primary endothelial cells. The BBBC also allowed for immunofluorescence imaging which helped confirm the formation and accuracy of the BBB. The results yielded that this BBBC model was effective for in vitro functional studies as drug screening for drugs that target or protect the BBB.

19Another idea that has gained immense popularity amongst organ chip researchers is the idea of a human-on-a-chip. This model aims to examine normal human physiology within a microfluidic system by combining single organ chip designs into a multi-organ chip design that allows the organs to work in conjunction with each other much like organs in the human body do. MOC and a complete human-on-a-chip design would allow for cheaper and more effective drug testing and thus would greatly benefit biomedical sectors.

20A revolutionary technique was carried-out that successfully manufactured a lung/liver-on-a-chip by connecting liver spheroid cultures with a 3D organotypic bronchial model. The experiment aimed to study the effect of certain aerosols on the lungs and incorporated the liver spheroid model to test the potential toxicity of the aerosols as well as their metabolites. The liver model was built using human HepaRG cells and the lung model was constructed with normal human bronchial epithelial cells. The study concluded that this MOC model was effective for demonstrating the assessment of compound toxicity on both the lungs and liver, and that it was relatively easy to use and maintain.

21Another experiment successfully fashioned a MOC that connected models of the GI tract and liver through the use of 3D cell culture. The 3D liver model was created by using a polymer scaffold in which a human hepatocellular carcinoma cell line (HepG2 C3A) was cultured on. The primary hIECs were derived from patients during a colonoscopy screening and cultured in a 3D matrigel culture. The GI and liver compartments of the chip were connected via gravity driven fluidic medium flow reducing the cost of the system dramatically since the purchase of a pump system was not necessary. The study concluded that this model is more accurate than other current in vitro models and contains the potential to eventually lead to personalized medicine as a result of the utilization of patient-derived cells.

22Although organ chip models have the potential to be useful tools to screen anticancer drug therapies, they require careful planning and precise execution. Organ chip models are still far from perfect and have improvements to be made. A challenge that exists in them today include them being more difficult to use than other 3D culture techniques. Another challenge has to do with how fragile some models can be; the complexity of the microsystems can cause experiments to be interrupted by something as small as the formation of a single bubble in the chamber. Lastly, cell structural integrity and functionality are often times limited in long-term experiments when using common media. As organ chip models improve and more accurate replicable organ models arise, there will be less of a need for animal models allowing for cheaper and more environmentally friendly drug screening processes.

23Understanding tumor characteristics by developing an accurate tumor model is the key to understanding the link between today’s various types of cancers. 3D tumor cells grown using 3D cell culture methods have claimed the spotlight in tumor cell biology research because of their innate ability to replicate the in vivo environment of a tumor cell in vitro. Although 2D cell culture techniques are still commonly used because of their convenience, their inability to mimic the pathophysiology of tumor cells often renders their use impractical due to their inaccurate response to radiation therapy and drugs. Cancer cell aggregates known as MCTS are grown using 3D culture methods via suspension or embedment in gels. MCTS grown using these methods allow for models that mimic the in vivo tumor microenvironments. MCTS can be grown via static suspension, hanging drop methods, magnetic levitation, spinner bioreactor, rotational bioreactor, microfluidic system, and gel embedding (; 20). These various methods allow for the replication of different microenvironments that can be found in specific types of tumors.

24Tumor models have also been used to study cellular signaling pathways in which cellular pathways can be mapped and compared to cells in a 2D cell culture model to determine if 2D models are viable or not. If the 2D model is unrepresentative of the 3D in vivo-like model, then researchers can assume the cellular signaling pathways in the 2D are inaccurate. Furthermore, 3D cancer models have also been used extensively in the study of gene expression. One study compared a 2D monolayer cell culture to a 3D cell culture in which 24 malignant and non-malignant breast cell lines were cultured generated by lrECM. Big discrepancies in gene expression were uncovered for genes encoding signal transduction proteins, leading researchers to conclude that cellular pathways vary between 2D and 3D cultures established on lrECM. Gene expression alterations were also discovered for malignant and non-malignant prostate cell lines when a similar test was conducted.

25Much like organ-on-a-chip models, tumor-on-a-chip models have gained increasing popularity for the same reason as organ chips. A glioblastoma tumor was grown on a chip using C6 cells and treated with magnetic hyperthermia therapy. After the cells were seeded in the 3D culture, magnetic nanoparticles were injected into the central cavity of the chip allowing them to come into contact with the 3D cell culture thus submitting them to an alternating magnetic field. A fluorescence assay was used to assess the efficacy of the magnetic hyperthermia treatment. The study concluded that all the tumor cells on the chip were lysed after 30 min of treatment. Although the study contained a limiting factor due to the lack of vascular network typically present in the tumor tissue, the study proved that organ chip methods of drug testing in glioblastomas hold high potential in future studies.

26A scrupulous investigation was conducted that aimed to mimic the progression of kidney cancer via a novel 3D metastatic cancer cell model. Previous metastatic cancer models have been created culturing cells in 3D but lacked the ability to interact with the correct physiopathological conditions as well as accurately reflect the effect of anticancer drugs in vivo. A 3D biomimetic liver microtissue modeled in DLM/GelMA hydrogel and subjected to continuous perfusion was used to culture the kidney cancer cells (Caki-1). This served as an effective model to mimic kidney cancer metastasis and discovered a linear anti-cancer correlation between the concentration of Caki-1 cells and the concentration of the drug 5-Fluorouracil.

27Immunotherapy is an area of research that has quickly gained popularity. Immunotherapy methods use the patients’ own immune system and either enhance the natural response to tumor antigens, or direct specific attacks on malignant cells. Typically, when 2D tumor cultures are treated with immunotherapy treatments, attrition rates are high due to the 2D model’s inability to replicate the three-dimensional complex characteristics of a tumor. In contrast, 3D cell cultures provide researchers with the ability to replicate the tumor model by successfully mimicking the 3D cell-matrix formation. This allows for the immune cells to attack the malignant target cells, much like an in vivo model would. Studies have shown that 3D-cultured tumor cells have greater resistance to cytotoxicity as a result of phenotypic changes that are not present in 2D-cultured tumor cells. A study conducted by Dangles-Marie et al. found that in 3D culture of a lung carcinoma cell line, there was a decrease in Hsp70, and thusly a decrease in antigen presentation. This decrease in antigen presentation caused cytotoxic T lymphocyte attacks to become less likely in the cells, making it evident that 3D tumor models resemble the in vivo tumor microenvironment more accurately than 2D tumor models.

28Due to recent success in treating melanoma skin cancer, researchers have begun modeling melanoma tumor cells in 3D culture spheroids to target molecular mechanisms aiding in resistance in current immunotherapy treatments. Melanoma spheroids are grown in vitro to model in vivo tumor cells by using juvenile primary fibroblasts and keratinocytes. Juvenile primary fibroblasts and keratinocytes can either be isolated from a sample of juvenile foreskin or can be purchased and are advantageous because they usually are less differentiated than adult primary skin cells. 3D melanoma spheroids are generated via the hanging drop method and provide researchers with the ability to study drug resistance within the cells. By using these methods, scientists are able to create tumor models on a patient-to-patient basis to test treatments, as well as develop general tumor models to test a variety of non-patient specific treatments.

29In an effort to understand how primary lung cancer progresses to metastatic lung cancer, one study used 3D cell culturing techniques to plot the migration of the cancer cells. Matrigel invasion assays were implemented on serum-starved cells in which non-invaded cells were removed after 24 h, and the chambers were stained with crystal violet to view the invaded cells. 3D invasion assays were also performed on HN12 cells and seeded in the SeedEZ 3D ring. The cells were stained with Texas-red phalloidin after 10 days of growth and viewed under a fluoresce microscope. What the study uncovered through the use of these methods, is that NAP1/NCKAP1 is highly correlated with primary NSCLC and metastasis relative to normal lung tissues. Furthermore, the overexpression of NAP1 causes MMP9 activation thus invoking invasion and metastasis. The usage of Matrigel and 3D invasion assays were crucial to understanding the link between NAP1 and MMP9 in order to help understand how primary lung cancer progresses to metastatic lung cancer.

30A novel 3D bone marrow niche model was assembled to study the effects of a new class of engineered immune cells known as TEGs (αβT cells engineered to express a defined γδTCR) on primary myeloma cells. TEGs proved their ability to migrate through the 3D culture as well as initiate a killing response directed at the primary myeloma cells. Prior to this experiment, no 2D models were effective in predicting the clinical success of a treatment highlighting the need for a patient-specific model supporting primary myeloma cells. Compared to the 2D models, the 3D model outperformed the 2D models with its ability to analyze specific homing as well as on- and off-target effects. With the help of 3D cell culture, this 3D bone marrow niche model allows studying novel immunotherapies, therapy resistance mechanisms, and possible side-effects of primary myeloma.

31crafted a 3D microfluidic model used to assess the impact monocytes have on TCR T cells in the hepatitis B virus (HBV). Previous studies have confirmed that monocytes interrupt natural T cell functions, but little is known about the effects monocytes have on TCR T cells. To test the efficacy of the 3D microfluidic model, the 3D model was compared to standard 2D assays when testing the effect of monocytes on TCR T cells. What they found, was that retrovirally transduced TCR T cell cytotoxicity toward cancer cells was suppressed while mRNA electroporated TCR T cell cytotoxicity was unaffected in the presence of monocytes in the 3D microfluidic model. In the standard 2D assay, however, the monocytes did not suppress cytotoxicity toward cancer cells in either the retrovirally transduced TCR T cells or mRNA electroporated TCR T cells. These data suggest that the 3D microfluidic model provides a more accurate assessment when investigating tumor-immune cell behavior and has the potential to uncover the impact of specific biological pathways on monocyte-TCR T cell interactions.

32When designing an in vitro model for a cell, it is imperative that the environment accurately represents a cell’s natural environment in vivo. One of the ways this is achieved is through proper TE techniques. TE was first introduced in 1988 at UCLA Symposia on Molecular and Cellular Biology by Professor Robert Nerem. When designing a tissue, the most important aspect of the tissue model is properly mimicking the porosity of the tissue in vivo. Among the various methods that exist, the methods commonly used for scaffold production in 3D cell cultures are freeze-drying, SCPL, electrospinning (ES), and 3D printing (3DP). As TE methodologies become more advanced, it may become feasible to construct entire organs as well as repair damaged organs using patient cells to avoid rejection from the patient’s body.

33Freeze-drying is a process that is used to create highly porous PGLA scaffolds. Through the homogenization of a polymer solution in an organic solution and water mixture, an emulsion is created. The emulsion is then rapidly cooled to keep it in a liquid state structure where freeze-drying is then implemented to remove the solvent and water ultimately maintaining the original 3D structure. Freeze-drying is an effective technique when making scaffolds with a porosity of more than 90% and a pore size anywhere from 20 to 200 μm (on average).

34Recent studies have shown that freeze-drying techniques used to create hydrogel scaffolds are effective in 3D cell culture. created a novel ready-to-use scaffold for cell culture with a hybrid of gelatin and polypropylene non-woven fabric via freeze-drying. The scaffold’s structural integrity was unchanged after over 90 days in storage, making it a good candidate for 3D cell culture because of its read-to-use capability. Furthermore, the addition of gelatin into the scaffold demonstrated an increase in porosity as well as liquid storage capability in 3D cell cultures.

35took a novel approach to fabricate hydrogels from yeast whole cell protein via freeze-drying. Being that some hydrogels can be difficult to consistently replicate and are often expensive to order, yeast protein hydrogels present a cheap and potentially reliable alternative for hydrogel fabrication. When freeze-drying yeast hydrogels, the pore size can be made as big as 100 μm and the hydrogels can absorb liquid up to 12 times their weight allowing cells to stay in a highly hydrated environment. Being that pore size between 5 and 350 μm is sufficient for 3D cell culture and adequate diffusion rates were observed in the hydrogel, yeast hydrogels fabricated via freeze-drying show tremendous potential for 3D cell culture.

36Solvent casting particulate leaching is a technique used to create porous scaffolds by mixing water-soluble salt particles into a biodegradable polymer solution and subsequently casting the mixture into the scaffold mold. The solvent is then removed via evaporation and the salt particles are leached out leaving behind a porous structure. SCPL is advantageous because it is relatively simple, and the pore sizes and porosity can be easily controlled by the size of the salt particles used and the salt/polymer ratio. The disadvantages, however, include limited interpore connectivity making uniform cell seeding and tissue growth difficult, residual salt particles being left over, and a thickness range of 0.5 to 2 mm for the scaffold as a result of soluble particles being difficult to remove from the interior of thick scaffolds.

37developed an enhanced SCPL technique that involves an extra step of centrifugation to create 45S5 BG reinforced PU scaffolds (PU-BG). PU-BG scaffolds were created using different centrifugal speeds of 1500 rpm, 2000 rpm, 2500 rpm, and 3000 rpm. The porosity and integrity of the PU-BG scaffolds were then compared to those made using the conventional SCPL method (no centrifugation). The scaffolds fabricated using the enhanced SCPL method contained a porosity of about 88% to 90% while the scaffolds created using the conventional SCPL method contained a porosity of about 81%. Furthermore, the scaffolds created via the enhanced SCPL method displayed high pore interconnectivity as a result of the centrifugation helping distribute the salt particles more evenly throughout the scaffold. As a result of centrifugation, however, the scaffolds created with the enhanced SCPL method displayed a lower compressive strength than the scaffolds made by conventional SCPL deeming them only capable for low load-bearing applications. Although scaffolds created via SCPL can be modified via the addition of a centrifugation step to have greater porosity and interconnectivity, the lack of compressive strength may pose difficulties when modeling bone tissue for bone tissue repair.

38developed SCPL polymer scaffolds to mimic the bone marrow niche so that medical therapies can be tested on cancers such as lymphomas and leukemias in vitro. A flexible PU polymer and a rigid PMMA polymer were compared when using NaCl as the porogen. The PU scaffold had a porosity of 91% and a compression of 29kPa, while the PMMA scaffold had a porosity of 84% and a compression of 1283kPa. Upon a collagen-coating, it was observed that human stromal HS-5 cells stuck to the scaffold supports as well as retained their pro-survival action toward co-cultured cancer cells avoiding the drug’s cytotoxic effect. These scaffolds are effective for mimicking the bone marrow microenvironment and have the potential to be extremely effective in preclinical drug studies.

393D printing has also allowed the printing of complex ECM-like scaffolds with such control that details can be fine-tuned at the micrometer level. A common method of printing scaffolds is a method known as electrospinning (ES). Electrospinning utilizes an electric field as a control mechanism to form and deposit polymer fibers onto a specific substrate. A charge imbalance is created upon the injection of an electrical potential into a melt or polymer solution. The Taylor cone is a stream of liquid produced at the critical point when the polymer solution is exposed to a high voltage causing it to become charged. The fibers are formed as the solvent evaporates off during the travel of the stream to the target. ES is among the most established TE techniques as electrospun collagen is commonly used to create tissue scaffolds due to how well it mimics the natural ECM.

40k When collagen is made, it is typically made with highly toxic organic solvents such as HFIP. designed a new way to create collagen scaffolds without the use of toxic organic solvents by using the sacrificing agent PVP instead of HFIP. By using the biodegradable synthetic polymer PLLCL as a base scaffold for the integration of collagen type I (Col), biomimetic PLLCL/PVP/Col hybrid scaffolds were created using co-electrospinning techniques. After ES was complete, the PVP was removed from the scaffold by being solubilized in water. To test whether the hybrid scaffold could support a 3D cell culture, NIH 3T3 mouse fibroblast cell line was cultured. The cells were successfully grown over a 14-day span and the results indicated that the hybrid PLLCL/Col scaffold promotes cellular adhesion and proliferation even during long-term studies much more effectively than standard 2D cell culture models.

41demonstrated a novel animal-friendly 3D electrospun polycaprolaceton (PCL) synthetic scaffold that effectively mimicked the collagen network of tissue. Human breast cancer cell lines JIMT-1 and MCF-7, the normal-like breast epithelial MCF-10A cell line, and mouse L929 fibroblasts were seeded in the 3D PCL scaffold and incubated for 7 days. After 7 days, the 3D cultures were analyzed using scanning electron microscopy, confocal laser scanning microscopy, and cryosectioning. The results showed that both the malignant as well as normal cell lines flourished in the 3D PCL scaffold indicating the potential to create a tumor ex vivo platform to screen therapeutic compounds.

42used ES in conjunction with photolithography to fabricate both nano- and micro-patterned PLGA/Collagen/nHAp fiber mats. 2D scaffolds and 3D scaffolds were used with MSCs to study the effect of geometric cues on proliferation and differentiation of MSCs. The MSC were seeded on the 2D scaffolds while MSC spheroids that were cultured for 3 days prior to seeding were seeded on the 3D scaffolds. The results indicated that higher osteogenic differentiation was found in the 3D spheroids than the 2D cells. This strategy of seeding 3D spheroids along with patterned substrates that resemble the natural tissue architecture may hold the power to help in regenerating a functional bone tissue.

43Stereolithography is another common method used to create artificial scaffolds. Stereolithography is a methodology of 3DP that prints an UV curable material in thin sheets layer-by-layer until the scaffold is complete. Each layer is laid on top of one another following the drying of the subsequent layer. After the scaffold is finished printing, it is placed under a UV light where it is postcured.

44fabricated a 3D model of the intestinal epithelium in vitro by combining a photopolymerizable hydrogel that promotes the growth of intestinal cell lines with stereolithography 3DP. Caco-2 intestinal epithelial cells were grown on the scaffold for 2 weeks and showed much higher rates of differentiation than standard 2D cultures. Thus, this model is a great candidate for studying intestinal homeostasis and regeneration mechanisms in vitro.

45combined stereolithography with electrospinning to create an advanced neural network scaffold. The inclusion of electrospinning fibers into the scaffold indicated significant improvements in neural stem adhesion when compared to 3D models without the added fibers. There were two types of fibers added to the 3D models: PCL fibers and PCL/gelatin fibers. What they discovered was the PCL/gelatin fibers enhanced the neural stem cell differentiation when compared to the PCL fibers without gelatin. The results of this study indicate that there is a high potential for developing unique 3D neural tissue models by implementing electrospinning and stereolithography techniques.

463D printing has allowed researchers to design microfluidic devices on the computer and then print them out using a 3D printer. Microfluidic devices are commonly made with polydimethylsiloxane but due to inconsistent reproducibility from lab-to-lab, some people have begun using 3DP to make them. Physiologically relevant dimensions can be reached within the channels of microfluidic devices via extrusion-based printing, stereolithography, and PolyJet. demonstrate two novel techniques to 3DP enclosed microfluidic channels via a PolyJet 3D printer. The first way implements a liquid to support cover layer prints while the second method uses a polycarbonate membrane to support the additional layers.

47designed an ECM collagen-based stretchable microfluidic system that resembles the in vivo blood vessels and allows for in vitro 3D cell culture. The ECM microfluidic channel was created using 3D printed water-soluble sacrificial molds. The stretchable design mimics the in vivo environment by allowing cells to be cultured in 3D while fluid shear stress and mechanical stretching occur simultaneously. This model could potentially be useful for studying vascular tissue formation due to its simple design and replicability.

483D cell culturing methods stand at the precipice of groundbreaking discovery and have the potential to unlock the answers researchers have been unable to uncover through the use of 2D cell culture techniques. With new technology however, comes obstacles and challenges. Although advantageous in many ways over 2D cultures, 3D culture tends to be more expensive and can be difficult to replicate cell microenvironments when using certain 3D culture methods. Furthermore, matrices often have multiple components that make them difficult to construct and require extensive amounts of labor.

49Imaging also becomes difficult when large scaffolds are used because there is a limit when scaling a single 3D format. Anchorage-dependent cultures such as hanging-drop plates and ultra-low-attachment plates can also be very difficult to image due to plate incompatibility with microscopes and uncentered spheroids in well plates. The most common way to analyze cellular phenotypes is by using conventional wide-field or confocal fluorescence microscopy. Fluorescence microscopy is often still challenging in 3D cell cultures because unlike 2D cell culture where only a single xy image is taken, 3D cell cultures must obtain a z stack by taking a series of xy images at fixed intervals in the vertical direction by automated microscopes. Having to take a series of xy images to obtain a z stack often increases the time significantly and as a result, higher magnification objectives (40–60×) are currently not practical for high-throughput setting as it takes too much time and storage space.

50Flow cytometry is a common technique used for 2D cell cultures to count cells, detect microorganisms, sort cells, detect biomarkers, detect protein engineering, and determine cell characteristics and functions. Flow cytometry has been used on 3D spheroids but requires the dissociation of the spheroids into a single-cell suspension via an enzyme such as trypsin and mechanical disruption. Because the spheroids must be broken up into a single-cell suspension, it ultimately becomes an endpoint assay as the cells are disposed following the completion of flow cytometry.

51Another common issue facing 3D cell culturing techniques is the automation of liquid handling. Liquid handling for suspension media and ultra-low-attachment can be easily automated, but more viscous liquids such as collagen- and Matrigel-containing hydrogels present unique challenges. Temperature sensitive polymerization in these gels requires quick liquid handling and careful environment control to avoid premature polymerization. Automation can often be achieved for many 3D culturing techniques in 96- or 384-well plates but further automation in miniaturized models may prove difficult as pipetting volumes are so small.

52A novel benchtop bioreactor was recently designed and tested in an article by. The bioreactor was built to allow the user to control the internal environment as well as use it in a small space outside of clean room environments. The bioreactor proved to be effective in supporting scaffold-based 3D progenitor cell cultures as well as presented the ability to provide solutions for automated cell therapy bioprocessing.

53Despite the limitations currently facing 3D cell culture methods, a survey from the HTS technologies found that two-thirds of people surveyed have plans to switch from 2D cell culture to 3D cell culture, with many of them having already switched. The more researchers who switch over to 3D, the quicker new methodologies will be developed that overcome the current limitations facing 3D cell culture. Likewise, many scientists already have plans for the future of 3D cell culture. The future of organoids remains bright, with the potential for developing alternative organ transplantation procedures as well as tumor models via patient-derived polypotent stem cells. Immunotherapy in 3D cell culture models is one of the most hopeful methods due to recent success relating to cancer treatment. As 3D tumor models become more advanced, immunotherapy treatments will advance to the point where more clinical trials can be performed with the potential of eventually finding a treatment for various cancers.

54Both 2D and 3D cell culture techniques provide methods which are necessary for advancing research. 3D cell culture, however, has proven it has the potential to completely change the way in which new drug treatments are tested, diseases are modeled, stem cells are utilized, and organs are transplanted. As 3D cell culture becomes more commonplace, the techniques will be better understood, and more advanced methods will arise. Researchers currently working to test new drug therapies via 2D cell culture models should seriously consider 3D cell culturing options. The benefits of co-culturing cells in 3D are superior to that of 2D cell culturing and as the techniques for tissue engineering improve, tumor models, cancer treatment therapies, and disease testing methodologies will improve.

55YT conceived the original idea. CJ wrote the manuscript with supervising from YT. Both authors provided critical feedback and helped to shape the manuscript.

56The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.