In vitro Characterization of Insulin−Producing β-Cell Spheroids

Yonela Ntamo, Ebrahim Samodien, Joleen Burger, Nolan Muller, Christo J. F. Muller and Nireshni Chellan

Published: 2021 January 28


Over the years, immortalized rodent β-cell lines such as RIN, HIT, MIN, βTC, and INS-1
have been used to investigate pancreatic β-cell physiology using conventional twodimensional (2D) culture techniques. However, physical and physiological limitations
inherent to 2D cell culture necessitates confirmatory follow up studies using sentient
animals. Three-dimensional (3D) culture models are gaining popularity for their
recapitulation of key features of in vivo organ physiology, and thus could pose as
potential surrogates for animal experiments. In this study, we aimed to develop and
characterize a rat insulinoma INS-1 3D spheroid model to compare with 2D monolayers
of the same cell line. Ultrastructural verification was done by transmission electron
microscopy and toluidine blue staining, which showed that both 2D monolayers and
3D spheroids contained highly granulated cells with ultrastructural features synonymous
with mature pancreatic β-cells, with increased prominence of these features observed in
3D spheroids. Viability, as assessed by cellular ATP quantification, size profiling and
glucose utilization, showed that our spheroids remained viable for the experimental
period of 30 days, compared to the limiting 5-day passage period of INS-1 monolayers.
In fact, increasing ATP content together with spheroid size was observed over time,
without adverse changes in glucose utilization. Additionally, β-cell function, assessed
by determining insulin and amylin secretion, showed that the 3D spheroids retained
glucose sensing and insulin secretory capability, that was more acute when compared
to 2D monolayer cultures. Thus, we were able to successfully demonstrate that our
in vitro INS-1 β-cell 3D spheroid model exhibits in vivo tissue-like structural features
with extended viability and lifespan. This offers enhanced predictive capacity of the
model in the study of metabolic disease, β-cell pathophysiology and the potential
treatment thereof.

Keywords: 3D culture spheroids, β-cell, transmission electron microscopy, insulin secretion, viability

FNDC5/irisin is expressed and regulated differently in human periodontal ligament cells, dental pulp stem cells and osteoblasts

Yang Yang, Helen Pullisaar, Maria A. Landin, Catherine Anne Heyward, Maria Schroder,
Tianxiang Geng
, Maria Grano and Janne Elin Reseland

Published: 2021 April



To examine the expression and regulation of fibronectin type III domain-containing protein 5/irisin (FNDC5/irisin) in primary human periodontal ligament (hPDL) cells, dental pulp stem cells (hDPCs) and osteoblasts (hOBs).


FNDC5/irisin was identified in sections of paraffin embedded rat maxillae, cryo-sections of 3D cultured spheroids hPDL cells, hDPCs and hOBs, 2D cultured hPDL cells, hDPCs and hOBs by immunohistochemistry. The expression of FNDC5/irisin was identified by qPCR, followed by sequencing of the qPCR product. Regulation of FNDC5/irisin expression in hPDL cells, hDPCs and hOBs were evaluated after administration of different concentrations of irisin and all-trans retinoic acid (ATRA). qPCR and ELISA were used to identify expression and secretion of FNDC5/irisin in odontoblast-like differentiation of hDPCs.


FNDC5/irisin was confirmed to be present in rat periodontium and dental pulp regions, as well as in 2D and 3D cultured hPDL cells, hDPCs and hOBs. BLAST analyses verified the generated nucleotide alignments matched human FNDC5/irisin. FNDC5/irisin gene expression was enhanced during odontoblast-like differentiation of hDPCs whereas the secretion of the protein was decreased compared to control. The protein signals in rat periodontal and pulpal tissues were higher than that of alveolar bone, and the expression of FNDC5/irisin was differently regulated by recombinant irisin and ATRA in hPDL cells and hDPCs compared to hOBs.


FNDC5/irisin expression was verified in rodent periodontium and dental pulp, and in hPDL cells, hDPCs and hOBs. The FNDC5/irisin expression was regulated by recombinant irisin and ATRA. Finally, expression and secretion of FNDC5/irisin were affected during odontoblast-like differentiation of hDPCs.

Keywords: FNDC5, Irisin, Human periodontal ligament cell, Human dental pulp stem cell, Human osteoblast

Anticancer Potential of Sutherlandia frutescens and Xysmalobium undulatum in LS180 Colorectal Cancer Mini-Tumors

Chrisna Gouws, Tanya Smit, Clarissa Willers, Hanna Svitina, Carlemi Calitz and Krzysztof Wrzesinski

Published: 2021 January 25


Colorectal cancer remains to be one of the leading causes of death worldwide, with millions of patients diagnosed each year. Although chemotherapeutic drugs are routinely used to treat cancer, these treatments have severe side effects. As a result, the use of herbal medicines has gained increasing popularity as a treatment for cancer. In this study, two South African medicinal plants widely used to treat various diseases, Sutherlandia frutescens and Xysmalobium undulatum, were evaluated for potential activity against colorectal cancer. This potential activity for the treatment of colorectal cancer was assessed relative to the known chemotherapeutic drug, paclitaxel. The cytotoxic activity was considered in an advanced three-dimensional (3D) sodium alginate encapsulated LS180 colorectal cancer functional spheroid model, cultured in clinostat-based rotating bioreactors. The LS180 cell mini-tumors were treated for 96 h with two concentrations of each of the crude aqueous extracts or paclitaxel. S. frutescens extract markedly decreased the soluble protein content, while decreasing ATP and AK per protein content to below detectable limits after only 24 h exposure. X. undulatum extract also decreased the soluble protein content, cell viability, and glucose consumption. The results suggested that the two phytomedicines have potential to become a source of new treatments against colorectal cancer.

Keywords: anticancer, colorectal cancer, functional spheroids, phytomedicine, sodium alginate, sutherlandia frutescens, three-dimensional cell cutlure, xysmalabium undulatum

Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicals

Martina Štampar, Helle Sedighi Frandsen, Adelina Rogowska Wrzesinska, Krzysztof Wrzesinski, Metka Filipič, Bojana Žegura
DOI: 10.1016/J.SCITOTENV.2020.143255
Published: 2021 February 10


The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations. 


Vitamin K2 Modulates Vitamin D‐Induced Mechanical Properties of Human 3D Bone Spheroids In Vitro

Maria Schröder, Elisabeth Aurstad Riksen,  Jianying He, Bjørn Helge Skallerud, Mona Elisabeth Møller, Aina‐Mari Lian, Unni Syversen, Janne Elin Reseland.
DOI: 10.1002/jbm4.10394
Published: 2020 July 14


Rotational culture promotes primary human osteoblasts (hOBs) to form three‐dimensional (3D) multicellular spheroids with bone tissue‐like structure without any scaffolding material. Cell‐based bone models enable us to investigate the effect of different agents on the mechanical strength of bone. Given that low dietary intake of both vitamin D and K is negatively associated with fracture risk, we aimed to assess the effect of these vitamins in this system. Osteospheres of hOBs were generated with menaquinone‐4 (MK‐4; 10μM) and 25‐hydroxyvitamin D3 [25(OH)D3; 0.01μM], alone and in combination, or without vitamins. The mechanical properties were tested by nanoindentation using a flat‐punch compression method, and the mineralized extracellular bone matrix was characterized by microscopy. The in vitro response of hOBs to MK‐4 and 25(OH)D3 was further evaluated in two‐dimensional (2D) cultures and in the 3D bone constructs applying gene expression analysis and multiplex immunoassays. Mechanical testing revealed that 25(OH)D3 induced a stiffer and MK‐4 a softer or more flexible osteosphere compared with control. Combined vitamin conditions induced the same flexibility as MK‐4 alone. Enhanced levels of periostin (p < 0.001) and altered distribution of collagen type I (COL‐1) were found in osteospheres supplemented with MK‐4. In contrast, 25(OH)D3 reduced COL‐1, both at the mRNA and protein levels, increased alkaline phosphatase, and stimulated mineral deposition in the osteospheres. With the two vitamins in combination, enhanced gene expression of periostin and COL‐1 was seen, as well as extended osteoid formation into the central region and increased mineral deposition all over the area. Moreover, we observed enhanced levels of osteocalcin in 2D and osteopontin in 3D cultures exposed to 25(OH)D3 alone and combined with MK‐4. In conclusion, the two vitamins seem to affect bone mechanical properties differently: vitamin D enhancing stiffness and K2 conveying flexibility to bone. These effects may translate to increased fracture resistance in vivo. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. 


Response to and recovery from treatment in human liver-mimetic clinostat spheroids: a model for assessing repeated-dose drug toxicity

Stephen J Fey, Barbara Korzeniowska, Krzysztof Wrzesinski.
DOI: 10.1093/toxres/tfaa033 
Published: 2020 June 12


Medicines are usually prescribed for repeated use over shorter or longer times. Unfortunately, repeated-dose animal toxicity studies do not correlate well with observations in man. As emphasized by the ‘3Rs’ and the desire to phase-out animal research, in vitro models are needed. One potential approach uses clinostat-cultured 3D HepG2–C3A liver-mimetic spheroids. They take 18 days to recover in vivo physiological functionality and reach a metabolic equilibrium, which is thereafter stable for a year. Acute and chronic repeated-dose studies of six drugs (amiodarone, diclofenac, metformin, phenformin, paracetamol and valproic acid) suggest that spheroids are more predictive of human in vivo toxicity than either 2D-cultured HepG2 cells or primary human hepatocytes. Repeated non-lethal treatment results in a clear response and return to equilibrium. Mitochondrial toxic compounds can be identified using a galactose-based medium. Some drugs induced a protective (or stress) response that intensifies after the second treatment. This 3D spheroid model is inexpensive, highly reproducible and well-suited for the determination of repeated-dose toxicity of compounds (naturally or chemically synthesized). 

Keywords: Drug toxicity, HepG2–C3A”>HepG2–C3A, recovery from treatment, repeated-dose, clinostat, 3D cell culture, acetaminophen, amiodarone, diclofenac, metformin, phenformin, valproic acid

Characterization of an Alginate Encapsulated LS180 Spheroid Model for Anti-colorectal Cancer Compound Screening

Tanya Smit, Carlemi Calitz, Clarissa Willers , Hanna Svitina, Josias Hamman, Stephen J Fey, Chrisna Gouws, Krzysztof Wrzesinski
Doi: 10.1021/acsmedchemlett.0c00076
Published: 2020 April 3


Colorectal cancer is one of the leading causes of cancer-related deaths. A main problem for its treatment is resistance to chemotherapy, requiring the development of new drugs. The success rate of new candidate cancer drugs in clinical trials remains dismal. Three-dimensional (3D) cell culture models have been proposed to bridge the current gap between in vitro chemotherapeutic studies and the human in vivo, due to shortcomings in the physiological relevance of the commonly used two-dimensional cell culture models. In this study, LS180 colorectal cancer cells were cultured as 3D sodium alginate encapsulated spheroids in clinostat bioreactors. Growth and viability were evaluated for 20 days to determine the ideal experimental window. The 3- (4,5- dimethylthiazol- 2- yl)-2,5-diphenyltetrazolium bromide assay was then used to establish half maximal inhibitory concentrations for the standard chemotherapeutic drug, paclitaxel. This concentration was used to further evaluate the established 3D model. During model characterization and evaluation soluble protein content, intracellular adenosine triphosphate levels, extracellular adenylate kinase, glucose consumption, and P-glycoprotein (P-gp) gene expression were measured. Use of the model for chemotherapeutic treatment screening was evaluated using two concentrations of paclitaxel, and treatment continued for 96 h. Paclitaxel caused a decrease in cell growth, viability, and glucose consumption in the model. Furthermore, relative expression of P-gp increased compared to the untreated control group. This is a typical resistance-producing change, seen in vivo and known to be a result of paclitaxel treatment. It was concluded that the LS180 sodium alginate encapsulated spheroid model could be used for testing new chemotherapeutic compounds for colorectal cancer. 


A sub-chronic Xysmalobium undulatum hepatotoxicity investigation in HepG2/C3A spheroid cultures compared to an in vivo model

Carlemi Calitz, Josias H. Hamman, Stephen J. Fey, Alvaro M. Viljoen, Chrisna Gouws and Krzysztof Wrzesinski
DOI: 10.1016/j.jep.2019.111897 
Published: 2019 April 19


Ethnopharmacology relevance: Traditional herbal medicines are utilized by 27 million South Africans. Xysmalobium undulatum (Uzara) is one of the most widely used traditional medicinal plants in Southern Africa. A false belief in the safety of herbal medicine may result in liver injury. Herb-induced liver injury (HILI) range from asymptomatic elevation of liver enzymes, to cirrhosis and in certain instances even acute liver failure. Various in vitro and in vivo models are available for the pre-clinical assessment of drug and herbal hepatotoxicity. However, more reliable and readily available in vitro models are needed, which are capable of bridging the gap between existing models and real human exposure. Three-dimensional (3D) spheroid cultures offer higher physiological relevance, overcoming many of the shortcomings of traditional two-dimensional cell cultures.

Aims of this study: This study investigated the hepatotoxic and anti-prolific effects of the crude X. undulatum aqueous extract during a sub-chronic study (21 days), in both a 3D HepG2/C3A spheroid model and the Sprague Dawley rat model.

Methods: HepG2/C3A spheroids were treated with a known hepatotoxin, valproic acid, and crude X. undulatum aqueous extract for 21 days with continuous evaluation of cell viability and proliferation. This was done by evaluating cell spheroid growth, intracellular adenosine triphosphate (ATP) levels and extracellular adenylate kinase (AK). Sprague Dawley rats were treated with the same compounds over 21 days, with evaluation of in vivo toxicity effects on serum chemistry.

Results: The results from the in vitro study clearly indicated hepatotoxic effects and possible liver damage following treatment with valproic acid, with associated growth inhibition, loss of cell viability and increased cytotoxicity as indicated by reduced intracellular ATP levels and increased AK levels. These results were supported by the increased in vivo levels of AST, ALT and LDH following treatment of the Sprague Dawley rats with valproic acid, indicative of hepatic cellular damage that may result in hepatotoxicity. The in vitro 3D spheroid model was also able to predict the potential concentration dependant hepatotoxicity of the crude X. undulatum aqueous extract. Similarly, the results obtained from the in vivo Sprague Dawley model indicated moderate hepatotoxic potential.

Conclusion: The data from both the 3D spheroid model and the Sprague Dawley model were able to indicate the potential concentration dependant hepatotoxicity of the crude X. undulatum aqueous extract. The results obtained from this study also confirmed the ability of the 3D spheroid model to effectively and reliably predict the long-term outcomes of possible hepatotoxicity.

Keywords: Toxicology, Herb induced liver injury, Drug induced liver injury, correlation between rats and spheroids  

Toxicity and anti-prolific properties of Xysmalobium undulatum water extract during short-term exposure to two-dimensional and three-dimensional spheroid cell cultures

Carlemi Calitz, Josias Hamman, Alvaro Viljoen, Stephen J. Fey, Krzysztof Wrzesinski and Chrisna Gouws
DOI: 10.1080/15376516.2018.1485805
Published: 2018 June 6


Xysmalobium undulatum (Uzara) is one of the most widely used indigenous traditional herbal remedies in Southern Africa. Commercially available Uzara plant material was used to prepare a crude aqueous extract, of which the toxicity potential was investigated in the hepatic HepG2/C3A cell line in both traditional two-dimensional (2D) and rotating three-dimensional (3D) spheroid cell cultures. These cultures were treated over a period of 4 days at concentrations of 200, 350, 500, and 750 mg/kg plant extract to protein content. Basic physiological parameters of the cell cultures were measured during exposure, including cell proliferation, glucose uptake, intracellular adenosine triphosphate levels, and adenylate kinase release. The results indicated that all physiological parameters monitored were affected in a dose dependent manner, with the highest concentration of Uzara crude water extract (750 mg/kg) resulting in toxicity. Anti-proliferating effects of Uzara crude water extract were observed in both the 2D and 3D cell cultures, with the most pronounced effects at concentrations of 350, 500, and 750 mg/kg. Discrepancies between results obtained from the 2D and 3D cell culture models may be attributed to the type of repair system that is initiated upon exposure, depending on where cells are within the cell cycle. DNA repair systems differ in cells within the G1 phase and non-diving cells, (i.e. cells found predominantly in in vitro 3D and the in vivo situation).

Keywords: Toxicology, Herb induced liver injury, Drug induced liver injury, correlation between 2D cell culture and spheroids

Metabolic Reprogramming and the Recovery of Physiological Functionality in 3D Cultures in Micro-Bioreactors

Krzysztof Wrzesinski and Stephen J. Fey
DOI: 10.3390/bioengineering5010022
Published: 2018 March 05


The recovery of physiological functionality, which is commonly seen in tissue mimetic three-dimensional (3D) cellular aggregates (organoids, spheroids, acini, etc.), has been observed in cells of many origins (primary tissues, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and immortal cell lines). This plurality and plasticity suggest that probably several basic principles promote this recovery process. The aim of this study was to identify these basic principles and describe how they are regulated so that they can be taken in consideration when micro-bioreactors are designed. Here, we provide evidence that one of these basic principles is hypoxia, which is a natural consequence of multicellular structures grown in microgravity cultures. Hypoxia drives a partial metabolic reprogramming to aerobic glycolysis and an increased anabolic synthesis. A second principle is the activation of cytoplasmic glutaminolysis for lipogenesis. Glutaminolysis is activated in the presence of hypo- or normo-glycaemic conditions and in turn is geared to the hexosamine pathway. The reducing power needed is produced in the pentose phosphate pathway, a prime function of glucose metabolism. Cytoskeletal reconstruction, histone modification, and the recovery of the physiological phenotype can all be traced to adaptive changes in the underlying cellular metabolism. These changes are coordinated by mTOR/Akt, p53 and non-canonical Wnt signaling pathways, while myc and NF-kB appear to be relatively inactive. Partial metabolic reprogramming to aerobic glycolysis, originally described by Warburg, is independent of the cell’s rate of proliferation, but is interwoven with the cells abilities to execute advanced functionality needed for replicating the tissues physiological performance.

keywords: bioreactors; 3D cell culture; spheroids; organoids; hypoxia; aerobic glycolysis; glutaminolysis; metabolic reprogramming; physiological performance; Warburg

Cell-free DNA in a three-dimensional spheroid cell culture model: A preliminary study

Janine AucampCarlemi CalitzAbel J.Bronkhorsta, Krzysztof Wrzesinskic, Sias Hamman,Chrisna Gouws, Piet J.Pretoriusa
Published: 2017 August


Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo state, may be of significant benefit for cfDNA research.

CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose consumption and was related to isolated cfDNA characteristics.

Changes in spheroid growth and stability were effectively mirrored by cfDNA characteristics. CfDNA characteristics correlated with that of previous two-dimensional (2D) cell culture and human plasma research.

3D spheroid cultures can serve as effective, simplified in vivo-simulating “closed-circuit” models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability.

Biological significance
3D cell cultures can be used to translate “closed-circuit” in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields.

Keywords: Cell-free DNA, 3D cell culture, Spheroid, Capillary electrophoresis, Rotating bioreactors

Recent advances in three-dimensional cell culturing to assess liver function and dysfunction: from a drug biotransformation and toxicity perspective

Carlemi Calitz, Josias H. Hamman, Stephen J. Fey, Krzysztof Wrzesinski & Chrisna Gouws
DOI: 10.1080/15376516.2017.1422580 


The liver is a vital organ fulfilling a central role in over 500 major metabolic functions, including serving as the most essential site for drug biotransformation. Dysfunction of the drug biotransformation processes may result in the exposure of the liver (and other organs) to hepatotoxins, potentially interacting with cellular constituents and causing toxicity and various lesions. Hepatotoxicity can be investigated on a tissue, cellular and molecular level by employing various in vivo and in vitro techniques, including novel three-dimensional (3 D) cell culturing methods. This paper reflects on the liver and its myriad of functions and the influence of drug biotransformation on liver dysfunction. Current in vivo and in vitro models used to study liver function and dysfunction is outlined, emphasizing their advantages and disadvantages. The advantages of novel in vitro 3 D cell culture models are discussed and the possibility of novel models to bridge the gap between in vitro and in vivo models is explained. Progression made in the field of cell culturing methods such as 3 D cell culturing techniques over the last decade promises to reduce the use of in vivo animal models in biotransformation and toxicological studies of the liver.

Keywords: 3D models; drug biotransformation; drug toxicity; in vitro models; liver dysfunction.

Acetaminophen-induced S-nitrosylation and S-sulfenylation signalling in 3D cultured hepatocarcinoma cell spheroids


Katarzyna Wojdyla, Krzysztof Wrzesinski, James Williamson,  Stephen J. Fey, Adelina Rogowska-Wrzesinska
DOI: 10.1039/C5TX00469A


Acetaminophen (APAP) is possibly the most widely used medication globally and yet little is known of its molecular effects at therapeutic doses. Using a novel approach, we have analysed the redox proteome of the hepatocellular cell line HepG2/C3A treated with therapeutic doses of APAP and quantitated both individual protein abundance and their reversible S-nitrosylation (SNO) and S-sulfenylation (SOH) modifications by mass spectrometry. APAP treatment results in a late, transient increase in ATP production and a multiplicity of alterations in protein abundance and modifications. The majority of the differentially SNO or SOH modified proteins are found in the endoplasmic reticulum and cytosol, suggesting that the source of reactive species is there. The cellular response indicates: constraint of fatty acid metabolism; reduction in ribosome construction and protein synthesis (to conserve ATP); maintenance of glutathione levels (by increased synthetic capacity); and an increased NADPH production (via the pentose phosphate pathway). This response appears to be coordinated, directly or indirectly, by the canonical Wnt and Nrf2 signalling pathways. Combined with the known role of NAPQI, these studies suggest that the physiological and toxicological responses form a continuum: therapeutic doses of APAP produce reactive species and NAPQI in the cytoplasm but result in little permanent damage. The cell mounts a multifaceted response which minimises disruption and repairs are effected within a day or two. Higher doses of APAP lead to intensified reactive species production, which increasingly disturbs mitochondrial function and eventually leads to cell death.

keywords: 3d model, hepatotoxicity, spheroids, proteomics, acetaminophen, toxicology, PTMs

From 2D to 3D – a new dimension for modelling the effect of natural products on human tissue

Krzysztof Wrzesinski and Stephen J. Fey
DOI: 10.2174/1381612821666151002114227

Keywords: Natural products, 3D cell culture, metabolic equilibrium, spheroids, bioreactor, physiology, toxicology, efficacy

Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns

Andrey Tvardovskiy, Krzysztof Wrzesinski, Simone Sidoli, Stephen J. Fey, Adelina Rogowska-Wrzesinska, Ole N. Jensen


Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.

Chromatin is a highly dynamic structure that must respond to different stimuli in order to orchestrate all DNA-dependent processes. Post translational modifications (PTMs)1 of histones play a major role in regulation of chromatin functionality. Evidence is emerging that not only ”classical” histone PTMs, such as methylation, acetylation, and phosphorylation at distinct residues, but also proteolytic processing of nucleosome proteins, known as “histone clipping,” can be involved in regulation of key cellular processes, such as transcriptional regulation, cell differentiation, and senescence (17).

Clipping of the histone H3 N-terminal tail was reported to be associated with gene activation in yeast. Santos-Rosa et al. demonstrated a serine protease activity in S. cerevisiae that cleaves histone H3 after residue Ala21 (A21) during sporulation and stationary phase (1). H3 clipping took place specifically within the promoters of sporulation-induced genes following the induction of transcription and prior to histone eviction from these DNA regions. Prevention of H3 N-tail cleavage by amino acid substitution at the endoproteinase recognition site (H3 Q19A, L20A) abolished expression of these genes, indicating that H3 clipping is essential for productive transcription.

The biological significance of histone clipping in higher eukaryotes is not yet understood but also appears to be related to functional commitment by the cell. Duncan et al. demonstrated that histone H3 is proteolytically cleaved by the enzyme Cathepsin L1 (CTSL1) at several sites between residues A21 and S28 during mouse ESC differentiation (5). The “in vitro” proteolytic activity of CTSL1 was found to be dependent on the H3 N-tail PTM status. H3K27me2 increased H3 cleavage by CTSL1, whereas H3K23ac reduced H3 cleavage. H3 clipping was also demonstrated in human ESCs (6).

A recent study by Duarte et al. proposed a role for H3 clipping in cellular senescence (7). Histone variant H3.3 was found to be proteolytically processed by CTSL1 upon oncogene-induced and replicative senescence in human fibroblasts and melanocytes. Ectopic expression of H3.3 and particularly its clipped proteoform was sufficient to induce senescence in fibroblasts presumably via transcriptional silencing of cell cycle regulatory genes.

Although the mechanism of regulation of histone clipping remains unclear, several studies suggested that this process might be affected by canonical histone PTMs (1, 3, 5, 8). However, because of technical challenges in the characterization of co-existing histone modifications the relation between histone clipping and covalent histone PTMs has remained poorly defined. In the present study, we address this question by using a middle-down proteomic workflow optimized in our laboratory for efficient characterization of combinatorial histone modifications (9).

First, we demonstrate that the N-terminal tails of two core histones, H2B and H3, undergo proteolytic processing in human hepatocytes both in vitro in hepatocarcinoma cell line HepG2/C3A and in vivo in primary hepatocytes and liver tissue. We find that cell culture conditions have profound effect on this process. Histone clipping takes place in HepG2/3CA cell line cultivated as a spheroid 3D culture (i.e. when cells are at their metabolic equilibrium (10)) but not when grown in a flat 2D culture using conventional cell culture techniques (when cells are in exponential growth). By using middle- and top-down proteomic approaches optimized for histone analysis we localize four different H3 cleavage sites and identify the position of H2B clipping. Finally we provide a comprehensive analysis of the PTM status of clipped H3 proteoforms and show that clipped H3 contain distinct PTM patterns enriched in K3K36 mono- and dimethylation.

Keywords: Hepatocytes, histone modification, tandem mass spectrometry, PTMs, chromatin alterations

The cultural divide: exponential growth in classical 2D and metabolic equilibrium in 3D environments

Krzysztof Wrzesinski, Adelina Rogowska-Wrzesinska, Rattiyaporn Kanlaya, Kamil Borkowski, Veit Schwämmle, Jie Daia, Kira E. Joensen, Katarzyne Wojdyla, Vasco Botelho Carvalho & Stephen J. Fey

Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different.

Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex.

We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance.

Keywords: 2d vs 3d cell culture, in vivo physiology, DNA alterations, Protein changes

Heteromer score – using internal standards to assess the quality of proteomic data

Adelina Rogowska-Wrzesinska, Krzysztof Wrzesinski and Stephen J. Fey
DOI: 10.1002/pmic.201300457


In the cell, the majority of proteins exist in complexes. Most of these complexes have a constant stoichiometry and thus can be used as internal standards. In this rapid communication, we show that it is possible to calculate a correlation coefficient that reflects the reproducibility of the analytical approach used. The abundance of one subunit in a heterodimer is plotted against the abundance of the other, and this is repeated for all subunits in all heteromers found in the data set. The correlation coefficient obtained (the “heteromer score”) is a new bioinformatic tool that is independent of the method used to collect the data, requires no special sample preparation and can be used retrospectively on old datasets. It can be used for quality control, to indicate when a change becomes significant or identify complexes whose stoichiometry has been perturbed during the experiment.

Keywords: Bioinformatics, Cell biology, Heterodimer, Heteromer score, Internal standard, Quality control

Microgravity spheroids as a reliable, long term tool for predictive toxicology

Stephen J. Fey and Krzysztof Wrzesinski
Doi: 10.1016/j.toxlet.2013.05.318


Spheroids grown in rotating ‘microgravity’ bioreactors (MC2 Biotek) from human C3A cells (an immortal hepatocellular carcinoma cell line) have been shown to need 18 days to reach maturity (as determined by their growth rate, viability and their production of urea, cholesterol and ATP). After this time they are metabolically stable for at least 24 days more; grow slowly (a doubling time of >20 days); produce physiological levels of urea, cholesterol and ATP; exhibit stable gene expression (for selected liver relevant genes); and can post translationally modify proteins in a
manner which mirrors those seen in vivo. Studies with 5 common drugs (acetaminophen, amiodarone, metformin, phenformin, and valproic acid) have shown that they are more predictive of lethally
toxic plasma levels in vivo than published studies using primary human hepatocytes. Shotgun proteomics has revealed that the gain of in vivo-like physiological properties is due to an exquisitely subtle modulation of the levels of hundreds of cellular proteins.

Keywords: Microgravity, in vivo functionality, 3D cell culture, toxicology, proteomics, correlation man vs. 2D vs. 3D

Determination of acute lethal and chronic lethal dose thresholds of Valproic acid using 3D spheroids constructed from the immortal human hepatocyte cell line HepG2/C3A

Stephen J. Fey and Krzysztof Wrzesinski
ISBN: 1624179525


Valproic acid (VPA) is a broad-spectrum antiepileptic drug that is now used commonly for several other neurological and psychiatric indications. While VPA is usually well tolerated, on rare occasions, it has been associated with severe, and sometimes, fatal liver injuries. These complications may also arise due to acute VPA overdose. As a branched chain carboxylic acid, VPA is readily metabolized in the liver via glucuronic acid conjugation, mitochondrial β- and cytosolic ω-oxidation to produce multiple metabolites, and it is probably some of these metabolites that are involved in its toxicity. While the actual mechanism of VPA hepatotoxicity is still not understood, two pathways have been in focus: the first involves the formation of reactive metabolites of VPA (and their subsequent covalent binding to cellular proteins) and the second is the development of oxidative stress in the cell. We describe here a culture system based on 3D spheroid culture of immortal hepatocytes which can determine the toxicity of valproic acid (or structurally or functionally related molecules) in vitro. The spheroids were used to follow changes in ATP production, glucose uptake and adenylate kinase following treatment and can be treated repeatedly with VPA to determine both acutely-lethal and chronically-lethal doses. This system is homeostatic and has been shown to be metabolically stable over at least 24 days and can therefore be used to follow recovery after treatment. 

Keywords: valproic acid, acute toxicity, 3d spheroids, hepatotoxicity, 

HepG2/C3A 3D spheroids exhibit stable physiological functionality for at least 24 days after recovering from trypsinisation

Krzystof Wrzesinski, Maria C. Magnone, Line Visby Hansen, Marianne Ehrhorn Kruse, Tobias Begauer, Maria Bobadilla, Marcel Gubler, Jacques Mizrahi, Christina Møller Andreasen, Kelan Zhang, Kira Eyed Joensen, Signe M. Andersen, Jacob Bastholm Olesen, Ove B. Schaffalitzky de Muckadel and Stephen J. Fey
DOI: 10.1039/C3TX20086H


Primary human hepatocytes are widely used as an in vitro system for the assessment of drug metabolism and toxicity. Nevertheless a cell system with higher stability of physiological functions is required for the investigation of drugs’ mode of action, pathway analyses and biomarkers evaluations. We recently discovered that the human hepatocellular carcinoma cell line, HepG2/C3A, cultured as spheroids in a 3D system can recover their main functions after trypsinisation within about 18 days. The objective of this study was to investigate whether the spheroids’ metabolic functions remained stable after this recovery period. Therefore we evaluated physiological capabilities of the spheroids (cell survival, growth rate, glycogenesis, ATP, cholesterol and urea synthesis and drug metabolism) and the expression of key genes related to the main liver pathways in spheroids cultured for an additional 24 days after full recovery (day 18). Here we show that after the recovery period, the 3D spheroid culture can provide a metabolically competent homeostatic cell model which is in equilibrium with its culture environment for more than 3 weeks. Such a stable system could be used for the assessment of the drugs’ mode of action, for biomarkers evaluation and for any systems biology studies which require medium- to long-term stability of metabolic functions.

Keywords: 3d cell culture, toxicology, Metabolic equlibrium in spheroids, regaining functionality, HEPG2 cells 

After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver

Krzystof Wrzesinski and Stephen J. Fey
DOI: 10.1039/C2TX20060K


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Keywords: 3d cell culture, spheroids, recover in vivo functionality, hepatocytes, trypsination effects

Determination of drug toxicity using 3D spheroids constructed from immortalized human hepatocytes

Stephen J. Fey and Krzysztof Wrzesinski
DOI: 10.1093/toxsci/kfs122
Published: 2012 March 27


Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this study was to investigate whether this observation could be extended to the determination of LD50 values and whether 3D data could be correlated to in vivo observations. We developed a noninvasive means to estimate the amount of protein present in a 3D spheroid from it is planar area (± 21%) so that a precise dose can be provided in a manner similar to in vivo studies. This avoided correction of the actual dose given based on a protein determination after treatment (when some cells may have lysed). Conversion of published in vitro LC50 data (mM) for six common drugs (acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid) to LD50 data (mg compound/mg cellular protein) showed that the variation in LD50 values was generally less than that suggested by the original LC50 data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented here) demonstrated similar LD50 values. Although in vitro 2D HepG2 data showed a poor correlation, the primary hepatocyte and 3D spheroid data resulted in a much higher degree of correlation with in vivo lethal blood plasma levels. These results corroborate that 3D hepatocyte cultures are significantly different from 2D cultures and are more representative of the liver in vivo.

Keywords: spheroid culture, drug toxicity, planimetry, protein-to-drug ratio, in vivo/in vitro correlation, LD50/LC50