**8. Yield and variability**

One easy way to initiate spheroid cultures is to use embryoid body plates (**Figure 9**).

Here, cells are centrifuged into the bottom of inverted square-based pyramid micro-indentations in microtitre plate wells. Not surprisingly, the spheroids are somewhat squarish immediately after their release from the embryoid body plate and there is quite a lot of loose cells (seen most clearly in the 'Day 0 and 2' image in **Figure 9**).

These loose cells do not sediment down as quickly as the spheroids and thus are lost during successive media changes. The remaining spheroids become steadily

*A Purpose-Built System for Culturing Cells as* In Vivo *Mimetic 3D Structures DOI: http://dx.doi.org/10.5772/intechopen.96091*

#### **Figure 9.**

*C3A spheroids at various times. C3A cells (800 cells per well) are left overnight in an embryoid body plate to form a cluster and are released on day 0 and then cultured in DMEM containing 5% foetal bovine serum in a non-humidified clinostat incubator at 37 °C and 5% CO₂/95% air for the number of days shown. The same magnification has been used for all images and the scale bar in the bottom corner illustrates 1 mm.*

rounder and more robust as they grow. Starting from a single embryoid body plate well, these procedures have been shown to produce large numbers of spheroids (about 1200) similar to those shown in **Figure 9** after 21 days (these would normally be cultured in four culture vessels). At this stage, each spheroid contains about 82,300 cells and contains 12.31 μg protein (C3A spheroids). The standard deviation of their diameters is less than 21% (after 21 days in culture) and this approach thus provides large numbers of very reproducible spheroids for further experimentation [39, 43].

Once C3A spheroids have recovered, they reach a metabolic equilibrium, characterised by a constant production of ATP, cholesterol and urea for at least 24 days [32]. During this period, treatment of these spheroids with for example any one of six commonly used drugs (acetaminophen (APAP), amiodarone, diclofenac, metformin, phenformin and valproic acid) causes them to respond (as shown by the increase in ATP production) and then recover to the pre-treatment conditions [43]. This can be repeated multiple times and has been proposed to be useful for assessing repeated-dose drug toxicity [39].

The liver is the primary source of many of the proteins found in the blood. To illustrate just how stable C3A spheroids are, they have been kept alive for 302 days. Even after this length of time C3A spheroids are quite capable of synthesising and post translationally modifying these blood proteins (**Figure 10**).

#### **Figure 10.**

*Proteins secreted from 302 day old C3A spheroids. Some of the proteins are named for reference: ACTB, actin beta; ALBU, albumen; APA4, apolipoprotein a I; APA4, apolipoprotein a IV; APOC3, apolipoprotein C III; APOE, apolipoprotein E, CO3 complement C3 alpha; CO4 complement C4; FETA Foetal albumen; FGL1, fibrinogen-like protein 1; FIBB, fibrinogen beta; FIBG, fibrinogen gamma; ITH4, inter alpha trypsin inhibitor heavy chain 4; MTN3, Matrilin3; PEDF, pigment epithelium derived factor; THRB, prothrombin; TRFE, Serotransferrin; TTHY, transthyretin. 302 day old C3A spheroids were biosynthetically labelled for 20 hrs with [35S]-methionine in order to be able to distinguish newly synthesised proteins from proteins present in the media. The whitish vertical streaks above ALBU indicate the presence of unlabelled BSA. Proteins in the growth media were collected by precipitation, washed, freeze dried, redissolved in lysis buffer and analysed by 2DGE according to [2]. Images were collected using AGFA phosphorimager plates and reader.*

### **9. Applications**

Already 8 years ago, clinostat spheroids constructed from C3A cells were shown to be more effective of predicting drug toxicity than primary human hepatocytes *in vitro* [43] and much of the data obtained since has been summarised from a biotransformation and toxicity perspective [44]. 3D spheroid cultures of primary human hepatocytes in chemically defined conditions have been used to evaluate the hepatotoxicity of 123 drugs in clinical drug induced liver injury (DILI) [45]. This has been followed by a demonstration that the system is a good candidate for determining repeated-dose toxicity (i.e. to detect accumulative toxicity) [39, 40].

C3A spheroids have also been used to reveal novel signalling pathways involved in drug treatment (acetaminophen) [46].

Currently one of the major weakness of testing for genotoxicity is the inability of indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds *in vitro*. C3A spheroids, cultivated in a clinostat system have been shown to express higher levels of these key metabolic enzymes from phase I and II, as well as DNA damage responsive genes. This suggests that this system can contribute significantly to a more reliable assessment of the genotoxic activities of both pure chemicals, and complex environmental samples. The system has been shown to be sensitive enough to detect genotoxicity even at the very low concentrations relevant to typical environmental exposure situations [47].

#### *A Purpose-Built System for Culturing Cells as* In Vivo *Mimetic 3D Structures DOI: http://dx.doi.org/10.5772/intechopen.96091*

Herbal medicines are often assumed to be safe because they are 'natural products', despite the lack of data. To reduce the cost and accelerate their testing, a C3A 3D spheroid model has been developed and benchmarked against Sprague Dawley rats to test one of the most widely used extracts, (*Xysmalobium undulatum*, commonly known as Uzara). The results showed comparable toxicological data [48].

Heteromeric proteins from spheroids even been used as an internal quality control for proteomic data [49].

Epigenetic marking and histone clipping have been shown to be recovered in spheroids [38] and this has been shown to occur during intestinal differentiation *in vivo* [50]. Metabolic reprogramming,which may be a key change during cancer development is also demonstrated in 3D spheroids [9]. The cell line LS180 is a very appropriate cell line for studying colorectal cancer, having been very gently developed but its application suffers from the fact that the cells do not readily form spheroids or organoids. This has recently been overcome by encapsulating the LS180 cells in sodium alginate. These spheroids were shown to respond to standard chemotherapeutic drug, paclitaxel at expected concentrations and even show the development of resistance often seen *in vivo* during paclitaxel treatment. The LS180 alginate spheroids are now being used to screen for novel chemotherapeutic compounds for colorectal cancer [51]. Similar studies have shown that pitavastatin can inhibit stem cell proliferation in colon carcinoma [52].

Spheroids and organoids are being used to investigate the self-organisation of multicellular tissues [53]. Human induced pluripotent stem cells (hiPS cells) have been used to generate neural spheroids that contain oligodendrocytes, neurons and astrocytes [28] and to mimic the blood brain barrier [54]. Primary and stem cells have been used to recapitulate the intricate pattern and functionality of pancreatic islets, working towards regenerative medicine for diabetes [55]. Progress is also being made towards producing transplantable photoreceptor precursors using pluripotent stem cell-derived retinal organoids to treat retinal degeneration diseases [56].

Bacterial-host interactions during Salmonella infections are being studied using iPSCs organoids and stem cell enteroids to mimic the intestinal villus and crypt [57].

Multicellular, physiologically active organotypic cultures are being use to study a wide variety of human viral pathogens with a view to pre-clinical evaluation of vaccines, antivirals and therapeutics [58].

Clinostat cultures are also being used in bone research. Low dietary intake of both vitamin D and K is negatively associated with fracture risk, often seen in persons suffering from osteoporosis. Treatment of primary human osteoblasts (hOBs) 3D multicellular spheroids with a combination of vitamin D and K, enhanced gene expression of periostin and collagen (COL-1), as well as inducing extended osteoid formation. The two vitamins apparently affected bone mechanical properties differently: vitamin D enhancing stiffness and K2 conveying flexibility to bone. It is anticipated that the combination of these effects may translate to increased fracture resistance *in vivo* [59].

#### **10. Conclusions**

Clinostat bioreactors systems clearly provide readily controllable 3D cell culture conditions, needing small amounts of cells, media or other compounds and provide sufficient cellular material for a wide variety of assays. The culture vessels and clinostat incubator described here, would be beneficial for many *in vitro* cellular models.

The advantages of culture stability for months, its reproducibility and the possibility to treat and then see the response and recovery (if necessary, for multiple times on the same culture) offer a great potential for future research. The novel equipment described here, will facilitate this research.

Furthermore, the fact that the clinostat system does not require changes in media, the use of scaffolds or special growth factors will not only facilitate the transition from other systems to this clinostat approach but will also allow the cells to respond in their own natural pre-programmed manner.

Thanks to 3D cell culture, the border between basic research and clinical applications is dissolving – and this new era of self-assembling tissue mimetic structures requires a new range of purpose-built equipment.
