Liver Toxicity: 3D Cell Culture Techniques

3D Cell Culture Techniques

Two-dimensional (2D) cell monolayer cultures are the mainstay of biomedical research, but they do not accurately reflect human physiology because they are not exposed to native stimuli including cell-to-cell and cell-matrix interacts. As a result, 2D cultures are far removed from original cells in organs of living organisms. Three-dimensional (3D) cell culture techniques including human organoids and organ-on-chip mimic the natural 3D environment, thus can be used to create highly predictive in vitro models for drug discovery, cancer research, and toxicity assessments.  3D cell cultures can help minimize the risk of adverse drug interactions by predicting a patient’s response and are useful for screening novel therapies in many disease areas including cancer, infectious diseases etc.1

The Need for Microfluidic Systems for Studying Live Toxicity 

Drug-induced liver injury (DILI) has resulted in the withdrawal of 14 approved therapies from the U.S. and European markets in the last three decades2,3. Assessing liver toxicity due to a new therapy is one of the required steps in the preclinical drug development. Although 2D cell cultures generate reproducible data, it is evident that they lack translation to human physiological systems4. Microfluidic liver models are a viable alternative for in vitro liver toxicity assessments and there is growing interest in this area. Also, In vivo solid tissues have a with strong dependence of perfusion of oxygenated blood, and In vitro improved oxygen delivery can be achieved with microfluidic systems that perfuse fresh media through 3D models. These 3D models recapitulate native liver tissue by including different cell types and the inclusion of a microfluidics perfusion system delivers oxygen and nutrients while removing waste products and metabolites. Thus, they have the potential to predict liver toxicity caused by a drug5 early in the development process, reducing the probability of hepatotoxicity in late-stage drug development or in clinical trials.

Toxicity Screening Using PerfusionPal

There are several organoid or organ-on-chip systems types that are useful for liver toxicity studies. For culturing cell lines, or primary cells or organoids, tools like PerfusionPal (Figure 1), provides perfusion of well-oxygenated media to the 3D scaffold in a user-friendly, multiplexed platform6.
 

Figure 1: PerfusionPal from Lena Biosciences provides nutrients and oxygen for cells via media perfusion.


Even more sophisticated in vitro models capturing the entire physiology of liver can be replicated in slices of actual liver7. Platforms like IVT LiveFlow facilitate media exchange or perfusion of liver slices in LiveBox1 or LiveBox2 chambers, respectively. LiveBox1 enables media flow over the tissue (Figure 2), while LiveBox2 can force the media to flow through a membrane supporting a slice of liver or a layer of hepatic cells.

 

Figure 2: LiveBox1


In LiveBox2 it is possible to culture different cell types on two sides of the membrane. In addition, multiple LiveBox chambers can be connected in series simulating production of drug metabolites on liver of in situ effect of other tissue types such as cortical cultures. Several MPS platforms support the co-culture of multiple liver cell types enabling delivery of oxygen and nutrients through perfusion with the choice dependent on user’s endpoints, biological and business requirements. MPS brings reproducible 3D biology with high translational value to established measurements of toxicity endpoints including CYP genes expression, cytotoxicity biomarkers (ATP, albumin, miR-122, alpha-GST), and metabolic readouts. The culture of human cells in the MPS platforms system can detect toxicity readouts that are not detectable in animal models, thus providing a more reliable workflow for the hepatotoxicity risk assessments.


References

  1. Edmondson R, et al. (2014). Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors. Assay Drug Dev Technol. doi: 10.1089/adt.2014.573
  2. 2.Zhou Y, Shen JX, Lauschke VM. (2019). Comprehensive Evaluation of Organotypic and Microphysiological Liver Models for Prediction of Drug-Induced Liver Injury. Front Pharmacol, 10:1093.
  3. 3.Bale SS, Vernetti L, Senutovitch N, Jindal R, Hegde M, Gough A, McCarty WJ, Bakan A, Bhushan A, Shun TY, Golberg I, De Biasio R, Usta BO, Taylor DL, and Yarmush ML. (2014). In vitro platforms for evaluating liver toxicity. Exp. Biol. Med. (Maywood), 239(9):1180–1191.
  4. 4.Watson DE, Hunziker R, Wikswo JP. (2017). Fitting tissue chips and microphysiological systems into the grand scheme of medicine, biology, pharmacology, and toxicology. Experimental Biology and Medicine, 242(16):1559–1572. doi:10.1177/1535370217732765
  5. Rudmann DG. (2019). The Emergence of Microphysiological Systems (Organs-on-chips) as Paradigm-changing Tools for Toxicologic Pathology. Toxicologic Pathology, 47(1): 4-10.
  6. Shoemaker JT, Zhang W, Atlas SI, Bryan RA, Inman SW, Vukasinovic J. (2020). A 3D Cell Culture Organ-on-a-Chip Platform With a Breathable Hemoglobin Analogue Augments and Extends Primary Human Hepatocyte Functions in vitro. Frontiers in molecular biosciences, 7: 568777. https://doi.org/10.3389/fmolb.2020.568777
  7. Soldatow VY, LeCluyse EL, Griffith LG, Rusyn I. (2013). In vitro models for liver toxicity testing. Toxicology Research, 2(1): 23–39.