Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids

A method has been developed for the quantitative evaluation of oxygen tension (PO2) distributions in multicellular spheroids measured with O2-sensitive microelectrodes. The experimental data showed that multicellular tumor spheroids in stirred growth media were characterized by a diffusion-depleted zone surrounding the spheroids. This zone was elicited by an unstirred layer of medium next to the spheroid leading to a continuous decrease in the PO2 values from the bulk medium towards the spheroid surface. Theoretical considerations demonstrate that the volume-related O2 consumption rate, Q, in the spheroids can be assessed by measuring the PO2 gradient in the diffusion-depleted zone outside the spheroids. Accordingly, Krogh's diffusion constant, KS, in the spheroids can be determined through measuring the PO2 gradient within the spheroids. The results obtained suggest that multicellular spheroids represent useful in vitro tumor models for the experimental and theoretical analysis of the interrelationship among O2 supply to tumor cells, O2 metabolism in tumors tissue, and the responsiveness of cancer cells to treatment.     

Indirect co-culture of lung carcinoma cells with hyperthermia-treated mesenchymal stem cells influences tumor spheroid growth in a collagen-based 3-dimensional microfluidic model

BackgroundMesenchymal stem cells (MSCs) have paradoxically been reported to exert either pro- or anti-tumor effects in vitro. Hyperthermia, in combination with chemotherapy, has tumor-inhibiting effects; however, its role, together with MSCs, so far is not well understood. Furthermore, a lot of research is conducted using conventional 2-dimensional in vitro models that do not mimic the actual tumor microenvironment.AimIn light of this fact, an indirect method of co-culturing human amniotic membrane-derived MSCs (AMMSCs) with collagen-encapsulated human lung carcinoma cells (A549) was performed using a 3-dimensional (3D) tumor-on-chip device.MethodsThe conditioned medium of AMMSCs (AMMSC-CM) or heat-treated AMMSCs (heat-AMMSC-CM) was utilized to create indirect co-culture conditions. Tumor spheroid growth characterization, immunocytochemistry and cytotoxicity assays, and anti-cancer peptide (P1) screening were performed to determine the effects of the conditioned medium.ResultsThe A549 cells cultured inside the 3D microfluidic chip developed into multicellular tumor spheroids over five days of culture. The AMMSC-CM, contrary to previous reports claiming its tumor-inhibiting potential, led to significant proliferation of tumor spheroids. Heat-AMMSC-CM led to reductions in both spheroid diameter and cell proliferation. The medium containing the P1 peptide was found to be the least cytotoxic to tumor spheroids in co-culture compared with the monoculture and heat-co-culture groups.ConclusionsHyperthermia, in combination with the anticancer peptide, exhibited highest cytotoxic effects. This study highlights the growing importance of 3D microfluidic tumor models for testing stem-cell-based and other anti-cancer therapies.     

Automated selective dissociation of cells from different regions of multicellular spheroids

Summary In this report we describe a new apparatus which has been developed for the automated selective dissociation of multicellular spheroids into fractions of viable cells from different locations in the spheroid. This device is based on the exposure of spheroids to a 0.25% solution of trypsin under carefully controlled conditions, such that the cells are released from the outer spheroid surface in successive layers. Study of the spheroid size, number of cells per spheroid, and sections through the spheroid with increasing exposure to trypsin demonstrate the effectiveness of this technique. The technique has been successfully used on spheroids from five different cell lines over a wide range of spheroid diameters. We also present data detailing the effect of varying the dissociation temperature, the mixing speed, the trypsin concentration, and the number of spheroids being dissociated. The new apparatus has several advantages over previous selective dissociation methods and other techniques for isolating cells from different regions in spheroids, including: a) precise control over dissociation conditions, improving reproducibility; b) short time to recover cell fractions; c) ability to isolate large numbers of cells from many different spheroid locations; d) use of common, inexpensive laboratory equipment; and e) easy adaptability to new cell lines or various spheroid sizes. Applications of this method are demonstrated, including the measurement of nutrient consumption rates, regrowth kinetics, and radiation survivals of cells from different spheroid regions. This work was supported by grants CA-36535, CA-22585, and RR-02845 from the National Institutes of Health, Bethesda, MD, the National Flow Cytometry Resource (NIH grant RR-01315), and by the Department of Energy, Washington, DC.     

A model for the growth of multicellular spheroids

Abstract. Based on biological observations and the basic physical properties of tri-dimensional structures, a mathematical expression is derived to relate the growth rate of multicellular spheroids to some easily measurable parameters. This model involves properties both of the individual cells and of the spheroid structure, such as the cell doubling time in monolayer, the rate of cell shedding from the spheroid and the depth of the external rim of cycling cells. The derived growth equation predicts a linear expansion of the spheroid diameter with time. The calculated growth rate for a number of spheroid cell types is in good agreement with experimental data. The model provides a simple and practical view of growth control in spheroids, and is further adapted to include parameters presumably responsible for the growth saturation in large spheroids.     

Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments

Adult rat hepatocytes formed floating multicellular spheroids in primary culture in an uncoated plastic dish with a positively charged surface. Cells in the spheroids formed in such a simple way were similar to those formed in dishes coated with proteoglycan fraction isolated from rat liver reticulin fibers; in both cases, cells maintained high ability to produce albumin and poor ability to proliferate in response to epidermal growth factor. Coating dishes with albumin was also helpful in spheroid formation; coating with 2-hydroxymethyl methacrylate resulted in formation of incomplete spheroids. Elimination of serum factors was essential for the formation of spheroids; when cells were washed with serum-containing medium before seeding or if the medium was replaced with a serum-containing medium, spheroid formation was completely inhibited. Collagens, fibronectin, and laminin, all of which promote the adhesion and spreading of hepatocytes on substrates, inhibited spheroid formation. Furthermore, collagens disintegrated spheroids, and cells in the monolayer initiated proliferation. Thus, two distinct, mutually exclusive features of primary culture of adult hepatocytes apparently exist; monolayer culture with proliferative activity in an adherent environment and spheroid culture with poor proliferative activity and high albumin-producing ability in a nonadherent environment.     

Recent advances in three-dimensional multicellular spheroid culture for biomedical research

Many types of mammalian cells can aggregate and differentiate into 3-D multicellular spheroids when cultured in suspension or a nonadhesive environment. Compared to conventional monolayer cultures, multicellular spheroids resemble real tissues better in terms of structural and functional properties. Multicellular spheroids formed by transformed cells are widely used as avascular tumor models for metastasis and invasion research and for therapeutic screening. Many primary or progenitor cells on the other hand, show significantly enhanced viability and functional performance when grown as spheroids. Multicellular spheroids in this aspect are ideal building units for tissue reconstruction. Here we review the current understanding of multicellular spheroid formation mechanisms, their biomedical applications, and recent advances in spheroid culture, manipulation, and analysis techniques.     

A novel method to prepare multicellular spheroids from varied cell types

A simple method for preparing multicellular spheroids from varied cell types has been successfully developed by using a stepwise gradient surface in cell attachability or detachability. The surface was composed of poly-N-isopropylacrylamide (PNIPAAm), a temperature responsive polymer, as a cell detaching component, and collagen as a cell attaching component. The surface functions as a culture substratum at 37 degrees C; then, when lowering the temperature of culture medium, the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature (LCST; about 30 degrees C), but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. The stepwise gradient surface which consisted of six different sectors was prepared by exposing a surface of the PNIPAAm-collagen mixture to ultraviolet (UV) irradiation six times using a photomask, sliding the hole position in the photomask, and changing the energy of UV irradiation. This was because crosslinking of collagen depended on the energy of UV irradiation, then, cell attachability to and detachability from the surface were tightly controlled by changing the energy.The stepwise gradient surface allowed us to easily determine optimal surface conditions to obtain good cell attachment and detachment as a self-supporting sheet from the surface to prepare multicellular spheroids. According to the evaluation of the attachability and detachability of 23 cell types, the optimal surface condition remarkably depended on each cell type. The detached cells under optimal surface conditions, including fibroblasts, osteoblastic cells, smooth muscle cells, and measangial cells, which were very difficult to form spherioids using conventional methods, were able to form multicellular spheroids. The results clearly demonstrate that the above-described method for preparing multicellular spheroids can be applied to varied cell types. (c) 1995 John Wiley & Sons, Inc.     

The growth and morphology of FRTL-5 thyroid epithelial cells grown as multicellular spheroids in vitro

Summary FRTL-5 cells, a diploid line of differentiated rat thyroid epithelial cells, have been grown as multicellular spheroids in spinner culture. Spheroids were initiated by seeding FRTL-5 cells either into Lab-Tek dishes or culture flasks with a 0.5% agar base. Thyroid stimulating hormone (TSH, >1.0 mU/ml) was required for initial cell aggregation and spheroid growth. After 1 wk cellular aggregates were transferred to suspension culture in spinner flasks. As with FRTL-5 monolayer cultures, continued spheroid growth required the addition of TSH to the culture medium. The most unique characteristic of the FRTL-5 spheroids was the development of central lumina similar to thyroid follicles in vivo. Follicular structures were absent from spheroids not stimulated with TSH. In the presence of TSH epithelial cells seem metabolically active with morphological evidence of biosynthesis of thyroglobulin-like material and basal laminar-like components. In contrast, all evidence of cellular metabolic activity is absent from cells in spheroids maintained in the absence of TSH. Thus, nontransformed FRTL-5 cells grown as three-dimensional multicellular spheroids responded to hormonal manipulation in a manner comparable to follicular epithelial cells in vivo. This spheroid model might therefore prove to be a very effective tool for investigating aspects of thyroid physiology and pathology in vitro. This work was supported by Grant CA-11198 and CA-20329 awarded by the National Institutes of Health, and a Biomedical Research Support Grant awarded to R. T. Mulcahy.     

Evaluation of the growth rate of MCF-7 breast cancer multicellular spheroids using three mathematical models

Abstract. Growth data on 60 multicellular spheroids of MCF-7 human breast cancer cells were fitted, on an individual basis, by the Gompertz, Bertalanffy and logistic equations. MCF-7 spheroids, initiated and grown in medium containing oestrogens, exhibited a growth rate that decreased continuously as spheroid size increased. Plots of spheroid volume v. time generated sigmoid curves that showed an early portion with an approximately exponential volume increase; a middle region or retardation phase characterized by a continuously decreasing growth rate; and, finally, a late segment or plateau phase approaching zero growth rate, that permitted an estimate of the maximum spheroid size (V_max). Growth curves generated by MCF-7 spheroids under different experimental conditions (hormones, drugs and radiation exposures) can be compared after normalization. Linearized forms of the fitted Gompertz curves provided a convenient way to express differences in growth rate.     

Efficient assembly of rat hepatocyte spheroids for tissue engineering applications

Freshly harvested primary rat hepatocytes cultivated as multicellular aggregates, or spheroids, have been observed to exhibit enhanced liver-specific function and differentiated morphology compared to cells cultured as monolayers. An efficient method of forming spheroids in spinner vessels is described. Within 24 h after inoculation, greater than 80% of inoculated cells formed spheroids. This efficiency was significantly greater than that reported previously for formation in stationary petri dishes. With a high specific oxygen uptake rate of 2.0 x 10(-9) mmol O(2)/cell/h, the oxygen supply is critical and should be monitored for successful formation. Throughout a 6-day culture period, spheroids assembled in spinner cultures maintained a high viability and produced albumin and urea at constant rates. Transmission electron microscopy indicated extensive cell-cell contacts and tight junctions between cells within spheroids. Microvilli-lined bile canaliculus-like channels were observed in the interior of spheroids and appeared to access the exterior through pores at the outer surface. Spheroids from spinner cultures exhibited at least the level of liver-specific activity as well as similar morphology and ultrastructure compared to spheroids formed in stationary petri dishes. Hepatocytes cultured as spheroids are potentially useful three-dimensional cell systems for application in a bioartificial liver device and for studying xenobiotic drug metabolism. (c) 1996 John Wiley & Sons, Inc.     

Migration and internalization of cells and polystyrene microspheres in tumor cell spheroids

The movement and internalization of ³H-labelled cells and of inert polystyrene microspheres within multicellular spheroids has been examined through histological sectioning and autoradiography. EMT6 and RIF-1 spheroids were cultured in spinner flasks for approx. 2.5 weeks. At this time, ³H-labelled cells and/or microspheres were allowed to adhere to the spheroid surface. Microspheres, ³H-labelled RIF-1 monolayer cells and ³H-labelled EMT6 monolayer cells were observed to move centripetally as a wave into EMT6 spheroids. In contrast, ³H-labelled trypsinized RIF-1 and EMT6 spheroid cells became mixed with the other non-labelled spheroid cells in homotypic RIF-1 and EMT6 spheroids, respectively. Reduction of spheroid growth by maintaining the spheroids at room temperature and by treatment with 2500 rads irradiation did not prohibit the internalization of ³H-labelled EMT6 cells and microspheres in EMT6 spheroids.     

Extended liver-specific functions of porcine hepatocyte spheroids entrapped in collagen gel

The potential use of porcine hepatocytes in a bioartificial liver device requires large quantities of viable and highly active cells. To facilitate the scaling up of the system, liver specific activities of hepatocytes should be maximized. One way of enhancing the specific activities is to cultivate hepatocytes as multicellular spheroids. Freshly isolated porcine hepatocytes form spheroids when cultivated in suspended cultures. These spheroids exhibit higher activities for a number of liver specific functions compared to hepatocytes cultivated as monolayers. However, these activities decreased in a few days in culture. Entrappment of spheroids in collagen gel sustained their metabolic activities at a stable level over 21 days. Production of albumin and urea by spheroid hepatocytes entrapped in collagen gels were 2 to 3 times higher than those by freshly isolated single cells. P-450 activity was demonstrated by metabolism of lidocaine to its main metabolite, monoethylglycinexylidide. Phase II drug metabolism was demonstrated by glucuronidation of 4-methylumbelliferone. This work shows that porcine hepatocyte spheroids entrapped in collagen maintain differentiated functions for an extended time period. Such hepatocyte spheroid entrappment system may facilitate the development of a bioartificial liver support device.     

In situ oxygen consumption rates of cells in V-79 multicellular spheroids during growth

The rate of consumption of oxygen by V-79 cells in multicellular spheroids was measured as a function of the spheroid diameter. In situ consumption was equal to that of exponentially growing cells for spheroids less than 200 micron in diameter. The rate of oxygen consumption decreased for cells in spheroids between 200 and 400 micron diameter to a value one-fourth the initial, then remained constant with further spheroid growth. Comparison of consumption rates for spheroid-derived cells before and after dissociation from the spheroid structure indicated that the spheroid microenvironment accounted for only 20% of the change in oxygen consumption rate. Cell-cell contact, cell packing, and cell volume were not critical parameters. Plateau-phase cells had a fivefold lower rate of oxygen consumption than exponential cells, and it is postulated that the spheroid quiescent cell population accounts for a large part of the intrinsic alteration in oxygen consumption of cells in spheroids. Some other mechanism must be involved in the regulation of cellular oxygen consumption in V-79 spheroids to account for the remainder of the reduction observed in this system.     

In vitro lung cancer multicellular tumor spheroid formation using a microfluidic device

The purpose of this study was to demonstrate self-organizing in vitro multicellular tumor spheroid (MCTS) formation in a microfluidic system and to observe the behavior of MCTSs under controlled microenvironment. The employed microfluidic system was designed for simple and effective formation of MCTSs by generating nutrient and oxygen gradients. The MCTSs were composed of cancer cells, vascular endothelial cells, and type I collagen matrix to mimic the in vivo tumor microenvironment (TME). Cell culture medium was perfused to the microfluidic device loaded with MCTSs by a passive fluidic pump at a constant flow rate. The dose response to an MMPs inhibitor was investigated to demonstrate the effects of biochemical substances. The result of long-term stability of MCTSs revealed that continuous perfusion of cell culture medium is one of the major factors for the successful MCTS formation. A continuous flow of cell culture medium in the in vitro TME greatly affected both the proliferation of cancer cells in the micro-wells and the sustainability of the endothelial cell-layer integrity in the lumen of microfluidic channels. Addition of MMP inhibitor to the cell culture medium improved the stability of the collagen matrix by preventing the detachment and shrinkage of the collagen matrix surrounding the MCTSs. In summary, the present constant flow assisted microfluidic system is highly advantageous for long-term observation of the MCTS generation, tumorous tissue formation process and drug responses. MCTS formation in a microfluidic system may serve as a potent tool for studying drug screening, tumorigenesis and metastasis.     

Effects of pre- and post-thaw cell-to-cell contact and trypsin on survival of freeze-thaw damaged mammalian cells

When multicellular spheroids, which simulate small bits of tissue, are exposed to a freeze-thaw (FT) cycle, the survival of the individual cells in the spheroid is higher if the cells of the spheroid are trypsinized and plated as single cells immediately after thawing than if the spheroid is allowed to remain intact for 4 hr and then trypsinized for plating. The results imply either that cell-to-cell contact inhibits repair of potentially lethal damage (PLD) or that accumulation of additional lethal or sublethal damage during the post-thaw period for cells in contact is taking place. Pre- and post-FT trypsinization of single cells indicate that trypsin does not enhance repair of PLD caused by a FT cycle.     

A reduction in the in situ rates of oxygen and glucose consumption of cells in EMT6/Ro spheroids during growth

The rates of consumption of oxygen and glucose by EMT6/Ro cells in multicellular spheroids were measured at various times during normal growth. In situ spheroid cellular consumption rates were similar to those of exponentially growing single cells up to a spheroid diameter of 150 micron. Further growth resulted in decreases in the rates of both oxygen and glucose consumption which were correlated with the increase in spheroid diameter and cell number. At a diameter of 1300 micron, both rates of cellular consumption had decreased by a factor of 2.5. The rates of consumption per unit of nonnecrotic spheroid volume decreased in a similar manner. Measurements with single cells demonstrated that the rate of oxygen consumption was coupled with glucose concentration, and vice versa. The rates of consumption for cells dissociated from small spheroids indicated that there was some effect of the spheroid environment. As the spheroids grew, however, association in the spheroid structure accounted for a smaller proportion of the total observed reduction in the rates of nutrient consumption. The presence of central necrosis also appeared to have no effect on the rates of consumption of these nutrients. Spheroid-derived cells showed a decrease in cell volume with growth as the cells accumulated in a quiescent state. Measurements with single cells demonstrated that oxygen and glucose consumption were correlated with cell volume and with the development of nonproliferating cells. We conclude that the observed decrease in oxygen and glucose consumption with growth in spheroids is largely due to the progressive accumulation of cells in a quiescent state characterized by an inherently lower cellular rate of nutrient utilization.     

Modeling mechanical inhomogeneities in small populations of proliferating monolayers and spheroids

Understanding the mechanical behavior of multicellular monolayers and spheroids is fundamental to tissue culture, organism development, and the early stages of tumor growth. Proliferating cells in monolayers and spheroids experience mechanical forces as they grow and divide and local inhomogeneities in the mechanical microenvironment can cause individual cells within the multicellular system to grow and divide at different rates. This differential growth, combined with cell division and reorganization, leads to residual stress. Multiple different modeling approaches have been taken to understand and predict the residual stresses that arise in growing multicellular systems, particularly tumor spheroids. Here, we show that by using a mechanically robust agent-based model constructed with the peridynamic framework, we gain a better understanding of residual stresses in multicellular systems as they grow from a single cell. In particular, we focus on small populations of cells (1–100 s) where population behavior is highly stochastic and prior investigation has been limited. We compare the average strain energy density of cells in monolayers and spheroids using different growth and division rules and find that, on average, cells in spheroids have a higher strain energy density than cells in monolayers. We also find that cells in the interior of a growing spheroid are, on average, in compression. Finally, we demonstrate the importance of accounting for stochastic fluctuations in the mechanical environment, particularly when the cellular response to mechanical cues is nonlinear. The results presented here serve as a starting point for both further investigation with agent-based models, and for the incorporation of major findings from agent-based models into continuum scale models when explicit representation of individual cells is not computationally feasible.     

Effect of Vascular Formed Endothelial Cell Network on the Invasive Capacity of Melanoma Using the In Vitro 3D Co-Culture Patterning Model

In vitro three dimensional (3D) cancer models were developed to observe the invasive capacity of melanoma cell spheroids co-cultured with the vascular-formed endothelial cell network. An array-like multicellular pattern of mouse melanoma cell line B16F1 was developed by magnetic cell labeling using a pin-holder device for allocation of magnetic force. When the B16F1 patterned together with a vascular network of human umbilical vein epithelial cells (HUVEC), spreading and progression were observed along the HUVEC network. The B16F1 cells over 80 µm distance from HUVEC remain in a compact spheroid shape, while B16F1 in the proximity of HUVEC aggressively changed their morphology and migrated. The mRNA expression levels of IL-6, MDR-1 and MMP-9 in B16F1 increased along with the distance the HUVEC network, and these expressions were increased by 5, 3 and 2-fold in the B16F1 close to HUVEC (within 80 µm distance) as compared to that far from HUVEC (over 80 µm distance). Our results clearly show that malignancy of tumor cells is enhanced in proximity to vascular endothelial cells and leads to intravasation.     

PIK3R3, part of the regulatory domain of PI3K,is upregulated in sarcoma stem-like cells and promotes invasion,migration, and chemotherapy resistance

To identify drivers of sarcoma cancer stem-like cells (CSCs), we compared gene expression using RNA sequencing between HT1080 fibrosarcoma and SK-LMS-1 leiomyosarcoma spheroids (which are enriched for CSCs) compared with the parent populations. The most overexpressed survival signaling-related gene in spheroids was phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), a regulatory subunit of PI3K, which functions in tumorigenesis and metastasis. In a human sarcoma microarray, PIK3R3 was also overexpressed by 4.1-fold compared with normal tissues. PIK3R3 inhibition using shRNA in the HT1080, SK-LMS-1, and DDLS8817 dedifferentiated liposarcoma in spheroids and in CD133+ cells (a CSC marker) reduced expression of CD133 and the stem cell factor Nanog and blocked spheroid formation by 61–71%. Mechanistic studies showed that in spheroid cells, PIK3R3 activated AKT and ERK signaling. Inhibition of PIK3R3, AKT, or ERK using shRNA or inhibitors decreased expression of Nanog, spheroid formation by 68–73%, and anchorage-independent growth by 76–91%. PIK3R3 or ERK1/2 inhibition similarly blocked sarcoma spheroid cell migration, invasion, secretion of MMP-2, xenograft invasion into adjacent normal tissue, and chemotherapy resistance. Together, these results show that signaling through the PIK3R3/ERK/Nanog axis promotes sarcoma CSC phenotypes such as migration, invasion, and chemotherapy resistance, and identify PIK3R3 as a potential therapeutic target in sarcoma.Subject terms: Cancer stem cells, Oncogenes, Sarcoma     

Differences in the uptake of transferrin bound 239Pu and 59Fe into multicellular spheroids of hepatocytes from adult male rats

Hepatocytes were cultured as monolayers and multicellular spheroids, respectively. The uptake of both transferrin-bound metals, iron and plutonium, differed significantly between these two culture systems. The uptake into the multicellular spheroids for plutonium was about 30 times greater, and for iron about 4 times greater, than in monolayer-cultured hepatocytes, which is not a consequence of proliferation and/or de-differentiation of the hepatocytes in the multicellular spheroid culture system. A comparison of the iron and plutonium uptake showed that plutonium was delivered to the cells to an 8-fold greater extent than iron if the hepatocytes were cultured as spheroids. Additionally, the binding of plutonium was not inhibited by preincubation of the spheroids with the iron-transferrin complex. Therefore, we propose that there are two different binding sites for iron and plutonium on hepatocyte membranes.