3D Cell Culture Tools and Techniques

• 2D vs 3D Cell Culture
  
• Advantages of 3D Cell Culture
• Overview of 3D Cell Culture Techniques
• Scaffold-based Techniques
• Scaffold-free Techniques

2D vs 3D cell culture techniques

Cell culture techniques are ubiquitous in areas of developmental biology, drug discovery, regenerative medicine and protein production. Since the introduction of cell culture techniques, cells have been cultured in two-dimensions, attached to tissue culture plasticware or ECM attachment proteins. Cells in the physiological environment have constant interaction with the extracellular matrix, regulating complex biological functions like cellular migration, apoptosis, transcriptional regulation, and receptor expression¹. In-vitro experimental data cannot be translated into clinical trials completely² when cells are grown in 2D conditions since complicated cellular signals between cells and its matrix cannot be reproduced³. Three-dimensional cell cultures address this challenge and serve as a better model representing in vivo physiological conditions closely. Table 1 indicates the difference between 2D and 3D cell culture systems.

Table 1. Cell behavior: 2D vs 3D cell culture conditions.

Key Characteristics	2D-Cell Culture	3D Cell Culture	References
Cell Shape	Flat and stretched	Natural shape (ellipsoid/polarized) is retained	10.3390/ijms160355173
Cell Interface to Medium	All cells are equally exposed to media components	As in physiological conditions, there is gradient availability of media components. Upper layer of cells are highly exposed over the lower layer (Heterogeneous exposure)	10.1016/j.semcancer.2005.05.0024, 10.1016/j.bbrc.2013.03.0085
Cell Junction	Cell junctions are less prevalent and does not resemble physiological conditions	Cell junctions are prevalent and enable cell to cell communication.	10.1371/journal.pone.00381476
Cell Differentiation	Moderately and poorly differentiated	Well differentiated	10.1016/j.yexcr.2012.09.0127
Drug Metabolism	Drug metabolism not well observed	Enhanced drug metabolism with increased expression of CYP enzymes	10.1007/s00204-013-1080-y8, 10.1089/ten.2006.12.13579
Drug Sensitivity	Cells are sensitive and drugs show high efficacy	Cells often show resistance and drugs show low potency	10.1111/j.1469-7580.2007.00778.x10
Cell Proliferation	Higher proliferation rate than in natural environment	Proliferation rate may be high or low, it is based on cell type and 3D-cell culture technique.	10.1007/s11626-997-0061-y11, 10.1016/j.biomaterials.2004.05.02812
Response to Stimuli	Poor response to mechanical stimuli of cells	well-established responses to mechanical stimuli of cells	10.1038/srep1955013
Viability	Sensitive to cytotoxin	Greater viability and less susceptible to external factor	10.1111/j.1469-7580.2007.00778.x10
Apoptosis	Highly susceptible to drug-induced apoptosis	Enhanced resistance to drug-induced apoptotic stimuli	10.3892/or_0000016714

Moreover, several studies have reported a difference in gene and protein expression profiles of the cells grown in 3D environment when compared to its 2D counterpart. Also, expression profiles in 3D culture conditions are thought to be more physiologically relevant than 2D cell culture conditions.

Advantages of 3D cell culture

Cellular events in 3D culture resemble physiological conditions closely and have the following distinct advantages over the 2D culture conditions

• Stem cells grown in 3D exhibit significantly higher differentiation potential¹⁵.
• Drug safety and efficacy studies are efficient and relatively easier to perform in 3D cultures reducing the time spent in drug discovery by pharmaceutical companies¹⁶. Drug-induced hepatotoxicity can be efficiently studied in 3D cell models¹⁶.
• 3D cultures provide better data in the prediction of drug resistance. Alkylating agents demonstrated resistance in a 3D culture that was comparative with in-vivo tumors¹⁷.
• Viral pathogenesis including viral growth, infection and pathogen-host interactions can be studied with reduced hazard levels using 3D models¹⁸.

Overview of 3D cell culture techniques

The choice of 3D cell culture technique should depend on several parameters, including the nature of the cells themselves (cell line, primary cell, tissue origin), or the final aim of the study. It’s crucial to evaluate these parameters before choosing the most relevant 3D cell culture technique.

Broadly, 3D cell culture techniques are classified as Scaffold-based or non-scaffold-based techniques.

Scaffold based techniques

In scaffold based techniques cells are grown in presence of a support. 2 major types of support can be used:

1. Hydrogel-based support: hydrogels are by definition polymer networks extensively swollen with water. Cells can be embedded in these hydrogels or simply coated at the surface. Depending on the nature of the polymer, hydrogels can be classified in to different categories (ECM protein-based hydrogels, natural hydrogels and synthetic hydrogels) with distinct properties.
2. Polymeric hard material based support: cells are cultivated in presence of fibers or sponge-like structures: cell recover a more physiological shape because they are not plated on a flat surface. Materials used for these supports can be polystyrene (adapted for imaging studies because of its transparency) but also biodegradable tools like polycaprolactone.
   
   Attributes related to these scaffolds are summarized in the following table:

Table 2. Scaffold-based 3D techniques overview with attributes.

Suitability: +++ = High; ++ = Medium; + = Low; - = Unsuitable; +/- = varies with scaffold components

Techniques Attributes	ECM-based Scaffolds	Natural Hydrogels	Synthetic Hydrogels	Polymeric Hard Scaffolds
Product Description	Laminin, Collagen, ECM Gel	Hystem®	TrueGel3D™, Hydromatrix™	3D Biotek Inserts, Cellusponge, Gelatin Scaffolds
Biological Relevance	+++	+++	+/-	+/-
Consistency/Reproducibility	-	++	+++	+++
Risk of Contamination	-	++	+++	+++
Modularity/Customization	-	+	++	-
Cell Retrieval	+/-	+	++	+++
Downstream Analysis (Imaging, Molecular Analysis)	+	++	++	++
HTS/HCS Analysis	+/-	+/-	+	-

Scaffold free techniques

Scaffold-free techniques allow the cells to self-assemble to form non-adherent cell aggregates called spheroids. Spheroids mimic the solid tissues by secreting their own extracellular matrix and displaying differential nutrient availability. Spheroids grown via non-scaffold based techniques are consistent in size and shape and are better in-vitro cellular models for high-throughput screening. Different platforms, from specialized plate to more integrated systems, can be used to generate spheroids: attributes of these techniques are described in the following table.

Application Note: Cancer Stem Cell Proliferation in Human Prostate and Breast Cancer Cell Lines Utilizing a New Defined Serum-Free 3D Spheroid Media

Protocol Guide: Cancer Stem Cell Tumorsphere Formation Protocol

Table 3. Scaffold-free 3D technique overview with attributes.

Suitability : +++ = High; ++ = Medium; + = Low; - = Unsuitable

Techniques Attributes	Low Attachment Microplates	Microfluidics Systems	Bioreactors
Product Description	Corning® Spheroids Plates	CellASIC ONIX Gradient Plate	Corning® Spinner Flask or Roller Bottles
Long Term Culture	++	+++	+++
Cell Retrieval	+++	-	+++
Image Analysis	+++	+++	-
Cost Effectiveness	++	-	-
HCS/HTS Analysis	+++	-	+

The evolution of 3D cell culture has the potential to bridge the gap between in vitro and in vivo experiments. The convenience of handling cells in vitro while obtaining results that reflect in vivo condition and avoiding ethical concerns of animal usage is making 3D cell culture techniques increasingly popular among researchers, but choosing the right system to develop a 3D cell culture model is not a trivial question.

The future will see the emerging of some more complex and advanced technologies like 3D bioprinting, an offshoot of 3D printing, helpful to print both biomaterials and living cells. 3D bioprinting has a wide medical application like skin grafting, which avoids a second wound site, characteristic of the traditional grafting methods. The major components for 3D bioprinting, like bio-inks, scaffold material, and biomaterials, are relatively well known to the scientific world. By configuring the order and position of these components various tissue products can be developed while simulating the physiological environment¹⁹. At the moment, the technique is in the early stage but has the potential to evolve as an indispensable tool for drug discovery and toxicity studies.

References

1. Kleinman HK, Philp D, Hoffman MP. 2003. Role of the extracellular matrix in morphogenesis. Current Opinion in Biotechnology. 14(5):526-532. https://doi.org/10.1016/j.copbio.2003.08.002

2. Hutchinson L, Kirk R. 2011. High drug attrition rates?where are we going wrong?. Nat Rev Clin Oncol. 8(4):189-190. https://doi.org/10.1038/nrclinonc.2011.34

3. Antoni D, Burckel H, Josset E, Noel G. Three-Dimensional Cell Culture: A Breakthrough in Vivo. IJMS. 16(12):5517-5527. https://doi.org/10.3390/ijms16035517

4. Kim JB. 2005. Three-dimensional tissue culture models in cancer biology. Seminars in Cancer Biology. 15(5):365-377. https://doi.org/10.1016/j.semcancer.2005.05.002

5. Yip D, Cho CH. 2013. A multicellular 3D heterospheroid model of liver tumor and stromal cells in collagen gel for anti-cancer drug testing. Biochemical and Biophysical Research Communications. 433(3):327-332. https://doi.org/10.1016/j.bbrc.2013.03.008

6. Soares CP, Midlej V, Oliveira MEWd, Benchimol M, Costa ML, Mermelstein C. 2D and 3D-Organized Cardiac Cells Shows Differences in Cellular Morphology, Adhesion Junctions, Presence of Myofibrils and Protein Expression. PLoS ONE. 7(5):e38147. https://doi.org/10.1371/journal.pone.0038147

7. Chitcholtan K, Asselin E, Parent S, Sykes PH, Evans JJ. 2013. Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer. Experimental Cell Research. 319(1):75-87. https://doi.org/10.1016/j.yexcr.2012.09.012

8. Schyschka L, Sánchez JJM, Wang Z, Burkhardt B, Müller-Vieira U, Zeilinger K, Bachmann A, Nadalin S, Damm G, Nussler AK. 2013. Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity. Arch Toxicol. 87(8):1581-1593. https://doi.org/10.1007/s00204-013-1080-y

9. Elkayam T, Amitay-Shaprut S, Dvir-Ginzberg M, Harel T, Cohen S. 2006. Enhancing the Drug Metabolism Activities of C3A? A Human Hepatocyte Cell Line?By Tissue Engineering Within Alginate Scaffolds. Tissue Engineering. 12(5):1357-1368. https://doi.org/10.1089/ten.2006.12.1357

10. Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge. J Anatomy. 0(0):070816212604002-???. https://doi.org/10.1111/j.1469-7580.2007.00778.x

11. Chopra V, Dinh TV, Hannigan EV. 1997. Three-dimensional endothelial-tumor epithelial cell interactions in human cervical cancers. In Vitro Cell.Dev.Biol.-Animal. 33(6):432-442. https://doi.org/10.1007/s11626-997-0061-y

12. Torisawa Y, Shiku H, Yasukawa T, Nishizawa M, Matsue T. 2005. Multi-channel 3-D cell culture device integrated on a silicon chip for anticancer drug sensitivity test. Biomaterials. 26(14):2165-2172. https://doi.org/10.1016/j.biomaterials.2004.05.028

13. Li Y, Huang G, Li M, Wang L, Elson EL, Jian Lu T, Genin GM, Xu F. 2016. An approach to quantifying 3D responses of cells to extreme strain. Sci Rep. 6(1): https://doi.org/10.1038/srep19550

14. Li. 1994. Survival advantages of multicellular spheroids vs. monolayers of HepG2 cells in vitro. Oncol Rep. https://doi.org/10.3892/or_00000167

15. Liu H, Roy K. 2005. Biomimetic Three-Dimensional Cultures Significantly Increase Hematopoietic Differentiation Efficacy of Embryonic Stem Cells. Tissue Engineering. 11(1-2):319-330. https://doi.org/10.1089/ten.2005.11.319

16. Meng Q. 2010. Three-dimensional culture of hepatocytes for prediction of drug-induced hepatotoxicity. Expert Opinion on Drug Metabolism & Toxicology. 6(6):733-746. https://doi.org/10.1517/17425251003674356

17. Kobayashi H, Man S, Graham CH, Kapitain SJ, Teicher BA, Kerbel RS. 1993. Acquired multicellular-mediated resistance to alkylating agents in cancer.. Proceedings of the National Academy of Sciences. 90(8):3294-3298. https://doi.org/10.1073/pnas.90.8.3294

18. Barrila J, Radtke AL, Crabbé A, Sarker SF, Herbst-Kralovetz MM, Ott CM, Nickerson CA. 2010. Organotypic 3D cell culture models: using the rotating wall vessel to study host?pathogen interactions. Nat Rev Microbiol. 8(11):791-801. https://doi.org/10.1038/nrmicro2423

19. Zhu W, Ma X, Gou M, Mei D, Zhang K, Chen S. 2016. 3D printing of functional biomaterials for tissue engineering. Current Opinion in Biotechnology. 40103-112. https://doi.org/10.1016/j.copbio.2016.03.014