A new technology allow print two-dimensional arrays of cells and proteins can mimic a variety of cellular environments in the body

Researchers have created a new technique that can rapidly “print’ two-dimensional arrays of cells and proteins that mimic a variety of cellular environments in the body.

Take a neural stem cell in the brain: Whether this cell remains a stem cell or differentiates into a fully formed brain cell is ultimately determined by a complex set of molecular messages the cell receives from countless neighbors.

Understanding these messages is key for scientists hoping to harness these stem cells to treat neurological conditions like Alzheimer’s or Parkinson’s.

With the help of photolithography and a creative use of programmable DNA, University of California, Berkeley, researchers have created a new technique that can rapidly “print” two-dimensional arrays of cells and proteins that mimic a wide variety of cellular environments in the body – be it the brain tissue surrounding a neural stem cell, the lining of the intestine or liver or the cellular configuration inside a tumor.

This technique could help scientists develop a better understanding of the complex cell-to-cell messaging that dictates a cell’s final fate, from neural stem cell differentiating into a brain cell to a tumor cell with the potential to metastasize to an embryonic stem cell becoming an organ cell.

“What’s really powerful about this platform is you can create in vitro tissues that capture the spatial organization of cells in the body, from the intestinal lining of your digestive tract to the arrangements of different cell types in the liver,” said Olivia Scheideler, who completed the research as a graduate student at Berkeley.

“I think you could apply this technique to recreate any tissue where you want to explore how cellular interactions contribute to tissue function.”

In a paper appearing today (Wednesday, March 18) in the journal Science Advances, Scheideler and her collaborators show that the new technique can be used to rapidly print intricate patterns of up to 10 different kinds cells or proteins onto a flat surface.

“Essentially, what this technique allows us to do is pattern different kinds of conditions in one shot and in a high-throughput manner,” said Lydia Sohn, Chancellor’s Professor of Mechanical Engineering at UC Berkeley and senior author of the paper.

“It provides a whole range of options for what you could study, because it’s so flexible. You can pattern many different kinds of cells or proteins.”

Caught on a DNA tether

In the new technique, each cell or protein is tethered to a substrate with a short string of DNA. While similar methods have been developed that attach tethered cells or proteins one by one, the new technique takes advantage of a patterning process called photolithography to attach, or print, each type of cell protein in one quick batch, greatly speeding up the process.

“It’s like color laser printing, where you print one color and then print another,” Sohn said.

Like photography, photolithography works by exposing a coated surface or substrate to a pattern of light, which initiates a chemical reaction that dissolves the coating in the illuminated areas, leaving a templated substrate.

In the new technique, the substrate is then bathed in strands of single-sided DNA, whose ends have been chemically altered to firmly latch on where the coating has been dissolved.

Each single-sided DNA strand is programmed have a specific sequence of the nucleotides adenine (A), thymine (T), guanine (G) and cytosine (C). Single-sided DNA strands with the complementary nucleotide sequence are embedded or attached to cells or proteins of interest.

Finally, the surface is washed with a mixture of cells or proteins attached to the complementary strands of single-sided DNA, which bond with the single-sided DNA already attached to the surface to form double-helix “tethers.”

“All the cells and proteins attach exactly where they should be because of the DNA programming,” Sohn said.

By repeating the process, up to 10 different kinds of cells or proteins can be tethered to the surface in an arbitrary pattern.

Conflicting messages

To demonstrate one of the many applications of the technique, Scheideler and co-author David Schaffer, Hubbard Howe Jr. Distinguished Professor of Biochemical Engineering at UC Berkeley, used the platform to study the chemical signaling that cues neural stem cells to differentiate into mature cells.

“Stem cells have programs embedded inside their DNA that tell them (to) either stay a stem cell or to differentiate into a mature cell,’” Schaffer said.

“And they receive a lot of information about what to do and which programs to activate from the environment, from other cells around them.

If we could learn how to make stem cells do our bidding, how to turn them into a particular cell type, then we could harness the stem cells to mass produce specialized cell types that were lost due to disease or injury.”

Neural stem cells in the brain regularly receive conflicting messages from their neighbors about how they should behave, Scheideler said.

One messenger, the FGF-2 protein, tells them to make more stem cells. The other, the ephrin-B2 protein, tells them to differentiate into a mature neuron.

In the new technique, cells and proteins are attached to a substrate via a DNA “tether.” The researchers used the technique to pattern neural stem cells alongside important cellular signaling proteins to find out how these proteins influence the cells’ ultimate fates: whether they remain stem cells or differentiate into mature neurons. The image is credited to Olivia Scheideler.

Scheideler used the new technique to pattern neural stem cells onto thousands of different arrays of the two proteins, FGF-2 and ephrin-B2, to see how the spatial organization of the two signals helps determine the cells’ ultimate fate.

She found that many stem cells differentiated into mature neurons, even when they were primarily in contact with FGF-2, or “stay a stem cell,” messengers.

When she looked closer, however, she found that those cells that differentiated were more likely to have small, finger-like extensions, or “neurites,” that touched the ephrin-B2 or “differentiate” messengers.

“The great thing about this patterning technology is you can easily replicate these little patterns hundreds or thousands of times across a slide,” Schaffer said.

“It is like running a thousand little independent experiments, each of which is a trial run to see how a stem cell listens to the cells around it. And then you can get very, very deep statistics about the various ways that it can be regulated.”

Co-authors of the paper include Chun Yang, Molly Kozminsky, Kira I. Mosher, Roberto Falcón-Banchs, Emma C. Ciminelli and Andrew W. Bremer of UC Berkeley and Sabrina A. Chern of Harvard University.

Funding: This research was supported by the National Institutes of Health through grants 1R01CA190843-01, 1R21EB019181-01A1, NIH/NCI F32 CA243354-01, and 1R21CA182375-01A1. It was also supported by the National Science Foundation Graduate Research Fellowship program, the Siebel Scholars program and a PEO Scholarship.

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Cancer is one of the most serious diseases causing almost one in six deaths globally, which is estimated to equal 9.6 million deaths in 2018[1]. Considerable efforts have been intended to develop effective approaches to cure cancer.

Among them, drug discovery could be one of the most important approaches aiming to identify and verify new and potent anticancer agents for both daily medication and chemotherapy.

For testing the capability of novel anticancer drugs, the experiments are performed on cell-based assays, which offer information about cellular responses to drugs in cost/time effective and high throughput manners.

Currently, two-dimensional (2D) platforms in which flat monolayer cells are cultured is still the most commonly used for the research of cell-based assays. The 2D cell culture systems are easy, convenient, cost-effective, and widely used. However, various drawbacks and limitations are still of concern.

The first drawback of a 2D cell culture systems is that an actual three-dimensional (3D) environment in which cancer cells reside in vivo is not accurately mimicked[2]. The irrelevant 2D environment may provide misleading results regarding the predicted responses of cancer cells to anticancer drugs[3].

Generally, standard preclinical screening procedures for therapeutic agents involve identification of compounds from the 2D cell culture system tests and animal model tests and then to the introduction of clinical trials[4].

Along with each phase, the percentage of efficient agents dramatically decreases. Less than 5% of anticancer agents and small molecule oncology therapeutics passed the clinical trials and were finally approved for marketing by the regulatory agencies[3].

One possible cause of the failure is that drug responses of 2D cell cultures systems did not consistently predict the outcome of clinical studies[5–7].

The key limitation of traditional 2D culture is the failure to imitate the in vivo architecture and microenvironments. As a consequence, there are many different features that 2D‑cultured cells possess compared with in vivo cells such as morphological characteristics, proliferation and differentiation potentials, interactions of cell-cell and cell-surrounding matrix, and signal transduction[8,9].

Such concerns inspired the emergence of 3D cell cultures systems, a promising approach to overcome the inconsistency between cell-based assays and clinical trials. The 3D cell culture systems provided the novel cell-based assays with more physiological relevance, especially the behavioral similarity to the in vivo cells.

Over the last decade, a variety of in vitro platforms was developed to achieve the 3D culture systems for cancer and stem cell applications such as novel drug development, cancer and stem cell biological research, tissue engineering for in vivo implantation, and other experimental cell analyses[10–12].

Thus, the study of cellular phenomena in a conditions that closely imitates in vivo scenery could be elaborately constructed in vitro[11,13,14].

Here, we aim to demonstrate the necessity of novel 3D cell culture systems and describe, compare, and contrast the 3D cell cultures techniques that has been developed to date. In addition, we also present the possibility to be applied in cancer and stem cell aspects.

TWO-DIMENSIONAL VS THREE-DIMENSIONAL CELL CULTURE MODELS

Cell culture is the most basic yet essential process for preclinical drug discovery. Even though the unreliable flaws of monolayer cell culture have been pointed out, 2D cell culture models are still the first option that scientists turn to due to its simplicity in order to obtain preliminary results. Nevertheless, 2D cultures may not sufficiently mimic the physiological conditions in a 3D network where in vivo cells reside. Therefore, deceptive data from 2D cell culture model often leads to the irrelevant prediction of drug efficacy and toxicity and finally causes the failure in drug validation and approval processes[23].

One obvious advantage of cell culturing in a 3D manner over 2D cell culture is that it contributes the expression of ECM components as well as the interactions between cell-cell and cell-matrix. The characteristics of 3D cell cultures and the traditional 2D cell culture models are shown in Figure 1.

The traditional 2D cell cultures result in a monolayer cell expanding on a flat surface of glass or commercial polystyrene plastic flasks for tissue culture (Figure 1A). In contrast, 3D cell cultures promote cells to form 3D spheroids by utilizing an ECM material (Figure 1B).

Cell spheroid is the important characteristic that resembles in vivo cells for further replicating cell differentiation, proliferation, and function in vitro.

Thus, 3D spheroid culture is considered an improved model for predictive in vitro cell-based assays and may deliver high physiological relevance for preclinical drug discovery, especially in cancer/stem cell research.

Generally, cells of multicellular organisms capable of forming tissues are in 3D arrangements with complex interactions within cell populations and also between cells and environments.

With the dynamics of nutrient and chemical transport between cells in the in vivo conditions, cells are hemostatically provided with a relatively constant supply of nutrients with the minimized level of waste products due to the activity of the circulatory system.

Therefore, the 3D arrangements of cells are the major employment for 3D cell culture with the optimal spatial organization of cells in the culture environment to be considered[24–26].

When cells are grown in 3D culture systems, cells also induce the formation of aggregates or spheroids within matrix or the culture medium. Even though with cell-cell interactions and cell-matrix interactions are not yet perfectly mimicked in a spheroid culture model, they are close enough to induce the morphological alteration of cells to not be relatively flat but closely resemble its natural shape in the body (Figure 1C).

Furthermore, within the spheroid structure, various stages of cells are established, including proliferating, quiescent, apoptotic, hypoxic, and necrotic cells due to the gradients of nutrients and oxygen level[27,28].

The proliferating cells could be found mainly at the outer layer of the spheroids because they are exposed to sufficient amounts of nutrients from the culture medium[29,30]. Cells at the core of spheroids tend to be in quiescent or hypoxic states because they are faced with the lack of oxygen, growth factors, and nutrients[31].

The cellular heterogeneity within a cell population is quite relevant to in vivo tissues, organs, and even tumors. At this point, due to cell morphology, interactions, and heterogeneity of cells grown in 3D culture, it is reasonable to hypothesize that the cellular processes of these cells are also applicable[32].

Comparisons of 3D spheroid culture models and 2D monolayer cell culture models were shown in Table ​Table1.1. Numerous studies have proven the differences in cell viability, morphology, proliferation, differentiation, cellular responses to stimuli, cell-cell communication, cell stiffness, migrant and invasive properties of tumor cells into surrounding tissues, angiogenesis stimulation and immune system evasion, drug responses, transcriptional and translational gene expression, general cell function, and in vivo relevance between cells cultured in 2D and 3D models.

For example, cell polarization could be more accurate depicted in 3D cell cultures models unlike in 2D models in which the cells can only be partially polarized. Moreover, greater stability and longer lifespans were found in 3D culture models; 3D spheroids can be cultured up to 3 wk, whereas 2D monolayer culture can last for less than a week due to the limitation of cell confluence[33].

Therefore, 3D cell culture models might be more appropriate for handling the long-term experiments and for determining long-term effects of the drug on cellular responses.

Table 1

Differences in two-dimensional vs three-dimensional cell culture models

Type of culture	2D	3D	Ref.
In vivo-like	Do not mimic the natural structure of the tissue or tumor mass	In vivo tissues and organs are in 3D form	Takai et al[102]
Proliferation	Tumor cells were grown in monolayer faster than in 3D spheroids	Similar to the situation in vivo	Lv et al[11]
Polarity	Partial polarization	More accurate depiction of cell polarization	Antoni et al[18]
Cell morphology	Sheet-like, flat, and stretched cells in monolayer	Form aggregate/spheroid structures	Breslin et al[103]
Stiffness	High stiffness (approximately 3 × 109 Pa)	Low stiffness (> 4000 Pa)	Krausz et al[104]
Cell-cell interaction	Limited cell-cell and cell-extracellular matrix interactions and no “niches”	In vivo-like, proper interactions of cell-cell and cell-extracellular matrix, environmental “niches” are created	Lv et al[11], Kang et al[105]
Gene/protein expression	Changes in gene expression, mRNA splicing, topology, and biochemistry of cells, often display differential gene/protein levels compared with in vivo models	Expression of genes and proteins in vivo is relevantly presented in 3D models	Bingel et al[92], Ravi et al[106]
Drug responses	Lack of correlation between 2D monolayer cell cultures and human tumors in drug testing.	Tumor cells in 3D culture showed drug resistance patterns similar to those observed in patients	Lv et al[11], Bingel et al[92]
The culture formation	From minutes to a few hours	From a few hours to a few days	Dai et al[33]
Quality of culture	High performance, reproducibility, long-term culture, easy to interpret, simplicity of culture	Worse performance and reproducibility, difficult to interpret, cultures are more difficult to carry out	Hickman et al[107]
Access to essential compounds	Unlimited access to oxygen, nutrients, metabolites, and signaling molecules (in contrast to in vivo)	Variable access to oxygen, nutrients, metabolites, and signaling molecules (similar to in vivo)	Pampaloni et al[108], Senkowski et al[30]
Cost during maintenance of a culture	Cheap, commercially available tests and media	More expensive, more time-consuming, fewer commercially available tests	Friedrich et al[35]

THREE-DIMENSIONAL CELL CULTURE TECHNOLOGIES

Because the advantages of 3D culture systems have become widely realized, there have been many studies intensively focused on the development and optimization of 3D cell culture technologies.

With the integration of the recent advances in cell biology, microfabrication techniques, and tissue engineering, a wide range of 3D cell culture platforms were constructed, including multicellular spheroid formation (liquid overlay culture and hanging drop method), hydrogel-based culture, bioreactor-based culture, bio-printing, and scaffold-based culture.

A summary of the advantages, disadvantages, and research stage of each model are shown in Table ​Table2.2. Although each 3D culture technique/platform are different in both principle and protocol, the same objectives that they share are to provide the similar features of in vivo cells in morphological, functional, and microenvironmental aspects. This section aims to briefly describe the key features of each technique.

Table 2

Proposed advantages, disadvantages, and research stage of different three-dimensional cell culture methods

Techniques	Advantages	Disadvantages	Research stage
Liquid overlay cultures and Hanging drops	(1) Easy-to-use protocol; (2) No added materials; (3) Consistent spheroid formation; control over size Co-culture ability; (4) Transparent; (5) High reproducibility; (6) Inexpensive; (7) Easy to image/harvest samples	(1) No support or porosity; (2) Limited flexibility; (3) Limited spheroid size; (4) Heterogeneity of cell lineage; (5) Lack of matrix interaction	(1) Basic research; (2) Drug discovery; (3) Personalized medicine
Hydrogel	(1) Large variety of natural or synthetic materials; (2) Customizable; (3) Co-culture possible; (4) Inexpensive; (5) High reproducibility	(1) Gelling mechanism; (2) Gel-to-gel variation and structural changes over time; (3) Undefined constituents in natural gels; (4) May not be transparent	(1) Basic research; (2) Drug discovery
Bioreactors	(1) Simple to culture cells; (2) Large-scale production easily achievable; (3) Motion of culture assists nutrient transport; (4) Spheroids produced are easily accessible	(1) Specialized equipment required; (2) No control over cell number/size of spheroid; (3) Cells possibly exposed to shear force in spinner flasks (may be problematic for sensitive cells)	(1) Basic research; (2) Tissue engineering; (3) Cell expansion
Scaffolds	(1) Large variety of materials possible for desired properties; (2) Customizable; (3) Co-cultures possible; (4) Medium cost	(1) Possible scaffold-to-scaffold variation; (2) May not be transparent; (3) Cell removal may be difficult	(1) Basic research; (2) Drug screening; (3) Drug discovery; (4) Cell expansion
3D bioprinting	(1) Custom-made architecture; (2) Chemical, physical gradients; (3) High-throughput production; (4) Co-culture ability	(1) Require expensive 3D bioprinting machine; (2) Challenges with cells/materials	(1) Cancer pathology; (2) Anticancer drug screening; (3) Cancer treatment; (4) Tissue engineering

Modified from Breslin et al[64]; Fang et al[47]; Leong et al[109].

Multicellular spheroids formation

Liquid overlay culture: Liquid overlay culture could be the simplest of all 3D cell culture Techniques (Figure ​2A). To create 3D culture models, the surface for cell culture is covered with a thin film of inert substrates, such as agar[34], agarose[35], or matrigel[36].

By preventing cell adhesion on the surface and providing the artificial matrix, liquid overlay culture easily promotes the aggregation of cells to become spheroids[37]. This technique is cost-effective and highly reproducible without requirement of any specific equipment[38]. Different cell types can be cocultured with this technique[39].

However, the number and size of formed spheroids are difficult to monitor[40]. Recently, ultra-low attachment plates have been developed and commercialized for the liquid overlay technique.

Such plates contain individual wells with a layer of hydrophilic polymer on the surface to overcome the requirement for manual coating, which prevents cell attachment. The specifically designed plates exhibit the capability to produce one spheroid per well and is favorable enough for medium-throughput applications[41].

Hanging drop technique: The hanging drop technique for 3D spheroid production was introduced by Johannes Holtfreter in 1944 for cultivating embryonic stem cells. The technique later became the foundation of scaffold-free 3D culture models capable of multicellular spheroid generation. Resulting spheroids could be generated with consistent size and shape controlled by adjusting the density of cell seeding.

As few as 50 cells up to 15000 cells could be varied to obtain the desirable size of spheroids[42]. In the very beginning, the hanging drop technique was carried out in the petri dish lid, by dropping a small volume of cell suspension (15-30 μL) with a specific cell density onto the lid.

Then, the lid was subsequently inverted and aliquots of cell suspension turned into hanging drops without dripping due to surface tension. Consequently, cells were forced to accumulate at the bottom tip of the drop, at the liquid-air interface, and further aggregate and proliferate until spheroids were formed (Figure 2B).

Recently, bioassay dishes have been used in place of petri dishes for more well-controlled experiments to facilitate the maintenance of moisture levels of the culture system, so that cell culture can be done in the same manner of standard cell culture procedures.

The hanging drop technique is relatively simple and applicable for numerous cell lines, and its reproducibility can be almost 100% for generating one 3D spheroid per drop[42]. The 3D spheroid obtained from this technique tends to be tightly packed rather than aggregated loosely, and low variability in sizes were observed. Kelm et al[42] reported that 3D spheroids exhibited patho/physiologically relevance because their structures were highly organized along with their produced ECM and turned to be a ‘tissue-like’ structure.

As this technique is based on the tendency of cells to aggregate to each other spontaneously instead of depending on the provided matrices or scaffolds, the problematic concerns regarding the effects from 3D structure formation are reduced.

However, the undeniable drawback of the hanging drop technique is the limited volume of the cell suspension. Only up to 50 µL of suspension, including the testing medium, can be accommodated onto the upside down surface unless dripping occurs as the surface tension is not enough to keep liquids attached on the surface[43]. Another limitation is the difficulty in changing culture medium during cultivation without disturbing the spheroids[31].

Hydrogels: Hydrogels are the networks of cross-linked polymeric material, which are generally composed of hydrophilic polymers with high water content (Figure ​2C)[44]. There are the swollen structures or microspheres integrated within the network for cell encapsulation and the circulation of nutrients and cellular waste in and out of the hydrogels[45].

Additionally, gels exhibit a soft tissue-like stiffness to potentially resemble natural ECM because they are made from mixtures of natural polymers such as collagen, and alginate, two of the most used substrates in 3D cell culture history[46].

The most common use of hydrogels is to be combined with a reconstituted basement membrane preparation extracted from mouse sarcoma, which has been commercialized by the Matrigel trademark (Corning Life Sciences, Tewksbury, MA, United States).

Even though such commercialized hydrogels are rich in ECM proteins, they also possessed some drawbacks, including the deficiency in gelation kinetic control, the undefined and uncontrollable polymer composition, and lack of mechanical integrity.

Lot-to-lot variability due to manufacturing mistakes and poorly defined composition also cause difficulty to determine the exact responses of cells to some particular stimuli[47].

Generally, hydrogels are fabricated based on both synthetic and natural polymers, which are water-absorbing, hydrophilic, and highly flexible materials. With the well-controlled fabrication processes and well-defined material composition, hydrogels have become the prominent materials for 3D scaffold development.

Because of their structural similarities to natural ECM, they are favorable for in vivo chemical delivery in a noninvasive manner[44]. A number of synthetic and natural materials can be incorporated into hydrogel formation, such as hyaluronic acids, polyethylene glycol[48], collagen, gelatin, fibrin, alginate, and agarose[49].

However, the natural hydrogels, like Matrigel and alginate gel, are considered to be more appropriate cell-encapsulated materials due to the great biocompatibility and mild gelling conditions.

The hydrogel technique for cell culture in a calcium alginate hydrogel was first developed by Lim et al[50] by mixing the cells with the alginate solution, then cross-linking and forming the hydrogel-based microspheres in an isotonic CaCl₂ solution (Figure ​2C).

The alginate hydrogels are very limited for cell adhesion, which is an advantage for cell encapsulation applications[51] that provide rapid, nontoxic, and versatile immobilization of cells within polymeric networks. In addition, the creation of artificial organs was also consolidated with encapsulating cells or tissue for the treatment of disease. The most well-known example was an artificial pancreas to be used in diabetes therapy[45].

The 3D cell culture can also be carried out in hydrogels and can be integrated with other cell culture models such as cell spheroid cultures, scaffold-based cell cultures, and microchip-based cell cultures[52].

Hydrogels are one potential technique to be used for 3D in vitro technology due to their biocompatibility, sufficient water content, and ECM-like mechanical properties[53]. Although hydrogels were not popularly applied to the field of drug screening, they have been widely used for the development of tissue engineering by mimicking cartilage, vascular, bone, and other tissues by mixing particular cells to hydrogel precursors before the gelling process in which cells are distributed evenly and homogeneously throughout the gels.

One reported case was the engineered cardiac tissues obtained from the neonatal rat cardiac myocyte culture in collagen hydrogels that were used for cyclic mechanical stretch research[54].

Hydrogels also facilitate the delivery of soluble or signaling molecules to cells and providing the supportive surroundings for cell growth and function. For example, transforming growth factor β was infused into polyethylene glycol hydrogels to govern the function of smooth muscle cells. In a similar manner, bone morphogenetic protein was covalently attached to alginate hydrogels to govern osteoblast migration and calcification[55].

Despite a variety of hydrogel type applications, Ca-alginate hydrogels are surely a potent candidate system for the delivery of cells to the infarcted heart because they are nontoxic, nonimmunogenic, do not facilitate pathogen transfer, and allow good exchange of waste products and nutrients[56,57].

Ca-alginate hydrogels were primarily implanted into the heart and shown not to induce harmful responses such as thrombosis[56] or fibrosis[58]. The gradual degradation, resulting from the dispersal of calcium crosslinks[59], generated nontoxic alginate polysaccharide degradation products, which can be excreted via urinary systems[60].

However, besides a number of advantages of hydrogels, the disadvantages of hydrogel are still present and should not be disregarded. The uncertainty and complexity in composition influenced by gelling mechanism may cause undesirable and nonspecific cellular responses. Additionally, pH based gelling mechanisms can negatively affect sensitive cells[52].

Bioreactors: Because the impact of 3D cell culture models as an appropriate in vitro laboratory platform for the discovery of therapeutics and anticancer agents have been concerned and drawn the attention of scientists, the crucial following step to cope with the increasing demand is the upscale 3D culture system from the laboratory to the industrial level. Bioreactors became the solution for great spheroid formation with a precise control system and guaranteed reproducibility[61].

With specifically designed 3D culture approaches, bioreactors have been adapted in many ways. For example, scaffolds have been added to the large cell culture chambers for high volume cell production.

Normally, a bioreactor for 3D spheroid production can be loosely classified into four categories: (1) Spinner flask bioreactors (Figure ​(Figure2D);2D); (2) Rotational culture systems; (3) Perfusion bioreactors; and (4) Mechanical force systems[18,62].

The general principle behind the bioreactor-based 3D culture systems is that a cell suspension with the optimal cell density is filled into the chamber with continuous agitation, either by gently stirring, rotating the chamber, or perfusing culture media through a scaffold using a pump system.

Bioreactors are equipped with media flowing systems to provide the nutrient circulation, metabolic waste expulsion, and homogeneity of the physical and chemical factors within the bioreactors. Therefore, bioreactor-based cell culture models are appropriate for intensive cell expansion and large-scale biomolecule production, such as antibodies or growth factors.

Although bioreactors are labor-intensive and capable of producing a large number of spheroids[63], the produced spheroids are still distributed heterogeneously in size and number of cell population[31]. Therefore, a manual spheroid selection is required for later replating onto a dish, if the spheroid size needs to be controlled[64].

Even though spheroid generation via bioreactors requires expensive instruments[65] and high quality/quantity of culture medium, the bioreactors can still provide greater advantages at the industrial level over other techniques[66].

Scaffolds: The 3D scaffolds are described as the synthetic 3D structures that are constructed from a wide-range of materials and possess different porosities, permeability, surface chemistries, and mechanical characteristics. They are mainly designed to mimic the in vivo ECM of the specific tissues for each particular cell type.

The 3D scaffold-based cell culture models have been applied to drug screening[11], drug discovery[47], and investigation of cell behaviors[47]. The 3D scaffolds are meant to be porous, biocompatible, and biodegradable, which provides appropriate microenvironments where cells naturally reside, supporting mechanical, physical, and biochemical requirements for cell growth and function[28].

Several biopolymers are used to generate porous scaffolds, which include collagen[11], gelatin[67], silk[68], chitosan[28], and alginate[28,69]. As such, various techniques have been used for the fabrication of scaffolds, such as gas foaming, freeze-drying, phase separation, solvent casting, and particulate leaching. Each technique results in different porosities, pore sizes and shapes, scaffold materials, and features. Among them, freeze-drying is considered the easiest technique to fabricate porous scaffolds[70].

Sequentially, natural or synthetic materials are polymerized, frozen, and freeze-dried. The frozen water embedded in the polymers is sublimated directly without going through the liquid phase resulting in a porous structure formation[71].

The freeze-drying technique for the fabrication of porous biodegradable scaffolds from polylactic and polyglycolic copolymer was first developed by Whang et al[72]. With such technique, the porosity and pore dimension of the scaffolds are varied depending on the various parameters such as the ratio of water and polymers and also the viscosity of polymer solution[73].

The porous alginate-based scaffolds can also be easily manufactured by a simple freeze-drying process (Figure ​(Figure2E).2E). However, it is difficult to generate pores with uniform diameter but can partially be controlled by varying the freezing temperature[74]. Another advantage of this technique is that no rinsing steps are required because dispersed water and polymer solution are removed directly via sublimation[72]. Additionally, the biodegradation rates of scaffolds are strongly dependent on polymer components and molecular weight[75].

To date, Ca-alginate copolymer is one of the most prominent materials for freeze-dried scaffolds. Several studies have used 3D Ca-alginate scaffolds as a cell culture platform for screening and efficacy testing of anticancer drugs and tissue engineering.

3D Ca-alginate scaffolds were proposed to allow more realistic cell phenomena, similar to those occurring in vivo during cancer formation and progression. Chen et al[69] developed a 3D porous Ca-alginate scaffold cell culture system combined with the functionally-closed process bioreactor to form bone-like tissue within the closely mimicked in vivo environments.

The Ca-alginate scaffolds were reported to support the growth and differentiation of human bone cell clusters, along with the upregulation of bone-related gene expression. Florczyk et al[28] developed chitosan-alginate scaffolds using the freeze-drying technique to study cancer stem cells transient behavior in vitro.

They found that 3D scaffold-based cultures of prostate, liver, and breast cancer cells exhibited reduced proliferation and tumor spheroid formation and increased expression of cancer stem-like cell associated mark genes (CD133 and NANOG) compared to 2D cell culture. Chitosan-alginate scaffolds were also observed to allow the efficient seeding of human umbilical cord mesenchymal stem cells, promoting the inhabitability of cells throughout the whole volume of the scaffold, which reflected good adhesion and proliferation[76].

3D bioprinting: 3D printing technique is a recently developed technology that, in general, is referred to as the construction of customized 3D structures under computational control in which materials are printed out, solidified, and connected together[51].

3D printing takes part in a wide-range application, including prototypic and industrial manufacturing, architecture, 3D art and design, and importantly, tissue engineering and regenerative medicine[77].

The 3D tissue printing that the biological constructs composed of cells and biomaterials are printed in a small dimension, ranging from several millimeters to a centimeter. The term is so called because the biocompatible materials, cells and supporting components are used to form a variety of 3D formats instead of any synthetic materials.

Therefore, cell function and viability can be sustained within the printed constructs (Figure ​2F)[77]. Various 3D bioprinting platforms can already generate vascular-like tubes[78], kidney[77], cartilage[79], artificial skin[80], and a wide range of stem cells including tissue constructs[81].

3D bioprinting is needed to precisely deposit cells, biomaterials, and biomolecules layer-by layer by computer-aided equipment and software, which has been possibly constructed by integration of modern science and technology knowledge, including cell biology, engineering, material science, and computer science[82].

By using alginate as the main biomaterial in a bio-ink, Zhao et al[83] studied the pathogenesis of cervical cancer using the developed cervical tumor model. Alginate, together with gelatin and fibrinogen, was mixed with HeLa cells to initiate gelation prior to printing and resemble the ECM components.

The printed constructs were later strengthened by the addition of a calcium chloride solution. Printed HeLa cells subsequently formed spheroids that exhibited more resistance to paclitaxel than 2D monolayer HeLa cells. Correspondingly, Dai et al[33] generated 3D bioprinted constructs of glioma stem cells using modified gelatin/alginate/fibrinogen biomaterials printed glioma stem cells. They could survive, proliferate, maintain the inherent characteristics of cancer stem cells, and exhibit differentiation and vascularization potential. In addition, their resistance against temozolomide were higher than those in the 2D cell culture model.

Besides the ability to generate geometric constructs containing viable cells, the 3D bioprinting technique also facilitated high throughput applications with precise reproducibility[84]. However, the main concerns are the requirement of the expensive 3D bioprinting machine and the negative effects on sensitive cells during the printing process. Cells could possibly be damaged due to osmotic, thermal, and mechanical stresses.

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Source:
UC Berkeley