3D Cell Culture

Advanced Translational Cell Research: A Reduction to Practice

  • Fig. 1: The design features of the Nano Well Plate (NWP) (A) An electron micrograph, showing the surface of 4 wells of the NWP, (B) A graphical representation of NWP with attached hydro gel based bioreactor system, (C) Photograph of the NWP showing wells and optical clarity of plastic substrate material and (D) Photograph of NWP and hydro gel bioreactor system in final assembly configuration.
  • Fig. 2: Differing growth patterns of cells maintained in the presence and absence of our Colloidal Suspension Medium. The upper and lower panels show representative images of cells cultured in the presence and absence of CSM. (A) 10X image of CHO-K-1 cells grown in media (- CSM), (B) 20X image of CHO-K1 cells grown in media (+ CSM), (C) 4X image of SKBR3 cells grown in media (- CSM), (D) 4X image of SKBR3 cells grown in media (+ CSM).
  • Fig. 3: Representative images of prostate cancer cells, which were incubated in 100 nL of CSM in Nano Well Plates over two days. Over this incubation period, cells aggregated into 3D structures.

The use of in vitro cell based assays has become commonplace in both academic and industrial research sectors. The acceptance of these has been largely due to their demonstrable utility as both drug-discovery and basic research tools. In recent times, we have observed a shift toward more biologically relevant approaches. Currently, the biggest driver in the field is to improve the relevance of cellular assays. To achieve this many are turning their attention to the use of primary cells and/or 3 dimensional cellular models. In this chapter we will discuss our current approach in this area.

The Importance of Miniaturizing Cell Based Assays for Translational Research
Assay miniaturization, especially when used in conjunction with technologies such as High Content Imaging (HCI), will permit a vast reduction in the number of cells required per experiment.  This is particularly important when conducting work on patient derived samples, such as those from biopsies, where the amount of usable biological material maybe extremely small [1, 2].  Indeed, we believe that a robust miniaturization approach is the most appropriate way to utilize rare and valuable primary material in cell based screens. These screens will be many times larger than currently possible.  To achieve this, we have developed a specialized Nano Well Plate (NWP) (fig. 1). This technology encompasses low profile 96 well micro-plates in which the well size can range from 500 to 700 microns in diameter and 300-800 microns in depth.  With this system, we have successfully performed experiments in assay volumes ranging from 50 – 200 nL [3].

Cell Viability in Low Media Volumes
To be viable for large-scale use, this technology has been developed with functionality comparable to a standard microtiter plate. The most striking feature the open architecture of the plate design is that it allows for free diffusion of respiratory gases and the rapid convenient dispensing of cells and experimental analyses. However, unlike many open architecture miniaturized devices, we have incorporated an on-board hydrogel based environmental buffering system (fig.

1), which has been designed to reduce harmful environmental fluctuations.

Two of the most important features that this technology offers are the ability to protect cells by (i) resisting rapid thermal fluctuations, and (ii) eliminating evaporation of media.  Our studies have demonstrated that a 100 nL droplet of water under standard laboratory conditions (without buffering) will evaporate completely within 60 seconds of dispensing. However, with our system a droplet of equivalent size can persist for many days.  Hence, without this means of controlling environmental conditions, performing assays at the nano-liter level is extremely difficult.

3 Dimensional Cell Based Assays for High Content Screening (HCS)
As mentioned above there is an increasing interest in adopting 3 dimensional cell based models as in vitro surrogates. These may offer potential benefits to both drug development and basic biological research programs. Indeed, in the field of cancer research the majority of studies involving cell based in vitro tumor models are currently performed using a 2D mono-layer format [4,5,6].  Despite their almost ubiquitous use in both academia and industry, it is becoming increasingly clear that cells cultured in a 2D format may not, in many cases, offer a representative model for tumors in vivo [7]. For example, it has been reported that there are many significant modifications in the behavior of cells grown in 2D compared with 3D [7].

These differences may largely be due to factors such as cell-cell communication, polarization and differentiation. Certainly in spheroidal 3D models, the cellular micro-environments (e.g. where gradients of oxygen and nutrients exist between the outer and inner regions of these structures ([8, 9]) are believed to lead to a stratification of cellular responses within the same sample [10]. Crucially, it is likely that the differences between 2D and 3D cell assay methods will have a significant impact on the way in which cells respond to drugs/treatments and could potentially result in misleading experimental data. To facilitate our translational studies in the field of cancer, we have undertaken to develop 3 dimensional assays for use within our automated high content imaging workflows. 

Early evaluation studies using technologies such as hanging drop and hydrogels revealed that many of the technologies currently available are not compatible with automation including high content imaging technologies [11]. To address these issues we have developed a new colloidal suspension medium (CSM), which has been specifically designed for use in automated and HCS workflows. This technology has the ability to keep cells in suspension but the liquid itself has a similar viscosity and density to standard supplemented culture media. These features make it very easy to use with automated liquid handling systems. Additionally, we have added the capability of deactivating the suspension agent, therefore allowing for quick and easy recovery of suspended cellular material.     

Proof of Principle Experiments
Our early work evaluating this technology was conducted using prostate cancer cell lines. Using these cells, we developed a methodology for growing tumor spheroids. As demonstrated in figure 2, cells cultured in the presence of our technology form spheroidal 3D structures. Those cultured in the absence of our technology do not form these structures and remain as 2D monolayers. Early studies also revealed that the prostate cells form spheroidal aggregates within 24 hours of initial seeding but then require 3-5 days to mature (fig. 3).  Additionally, high content studies demonstrated that by using this system we were able to observe significant changes in spheroidal size in 3D cultures treated with cytotoxic drugs (data not shown). Furthermore, the expression of a prostate cancer biomarker was elevated in cells cultured in 3D compared with 2D.

Future Directions
The ultimate goal is to combine these novel technologies, thus creating an automatable HCS assay format. We believe this will, for the first time, make possible the utilization of rare and valuable cell types (such as those derived from primary sources) for screens of magnitudes that are currently out of reach of more conventional available technologies. As shown in figure 3, we have already completed proof of principle experiments where tumor spheroids have been cultured from just a few hundred cells in volumes of approximately 100 nL. This article was adapted from the European Pharmaceutical Review Volume 18 issue 3 2013.

[1]    Bailey S.N. et al.: Drug Discovery Today 7 (18 Suppl) S113–118 (2002)
[2]    Giuliano K.A. et al.: Assay and Drug Development Technologies 3 (5) 501–514 (2005)
[3]    Davies A. et al.: G.I.T. Laboratory Journal Europe 7–8 (2011)
[4]    Box C. et al.: Semin Cancer Biol. 20 (3) 128–138 (2010)
[5]    Desgrosellier J.S. and Cheresh DA.: Nat Rev Cancer. 10 (1) 9–22 (2010)
[6]    Joyce J.A. and Pollard J.W.: Nat Rev Cancer. 9 (4) 239–252 (2009)
[7]    Friedrich J. et al.: Int J Radiat Biol. 83 (11-12) 849–71 (2007)
[8]    Acker H. et al.: British Journal of Cancer. 56, 325–327 (1987)
[9]    Carlsson J. and Acker H.: International Journal of Cancer 42, 715–720 (1988)
[10]    Ivascu A. and Kubbies M.: J Biomol Screen. 11 (8) 922–32 (2006)
[11]    3D Cell Culture Trends 2010 Report, published by HTStec limited, Cambridge, UK, February (2010)

Dr. Anthony Mitchell Davies
Sarah-Louise Ryan
Anne-Marie Baird, PhD
Aaron James Urquart, PhD

Queensland University of Technology
Institute of Health and Biomedical Innovation
Translational Research Institute
Woolloongabba, Brisbane, Australia

Prof. Derek J Richard
Prof. Ken O’Byrne

Queensland University of Technology
Institute of Health and Biomedical Innovation
Cancer and Ageing Research Program
Woolloongabba, Brisbane, Australia

Alice Vajda, PhD
Prof. Laure Marignol, PhD

Institute of Molecular Medicine
Trinity College Dublin
Dublin, Ireland

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