Numerical studies of continuous nutrient delivery for tumour spheroid culture in a microchannel by electrokinetically-induced pressure-driven flow

Continuous nutrient delivery to cells by pressure-driven flow is desirable for cell culture in lab-on-a-chip devices. An innovative method is proposed to generate an induced pressure-driven flow by using an electrokinetically-driven pump in a H-shape microchannel. A three-dimensional numerical model is developed to study the effectiveness of the proposed mechanism. It is shown that the average velocity of the generated pressure-driven flow is linearly dependent on the applied voltage. Considering the culture of a multicellular tumour spheroid (MTS) in such a microfluidic system, numerical simulations based on EMT6/Ro tumour cells is performed to find the effects of the nutrient distribution (oxygen and glucose), bulk velocity and channel size on the cell growth. Using an empirical formula, the growth of the tumour cell is studied. For low nutrient concentrations and low speed flows, it is found that the MTS grows faster in larger channels. It is also shown that, for low nutrient concentrations, a higher bulk liquid velocity provide better environment for MTS to grow. For lower velocities, it is found that the local MTS growth along the flow direction deviates from the average growth.     

Microfluidic device flow field characterization around tumor spheroids with tunable necrosis produced in an optimized off-chip process

Tumor spheroids are a 3-D tumor model that holds promise for testing cancer therapies in vitro using microfluidic devices. Tailoring the properties of a tumor spheroid is critical for evaluating therapies over a broad range of possible indications. Using human colon cancer cells (HCT-116), we demonstrate controlled tumor spheroid growth rates by varying the number of cells initially seeded into microwell chambers. The presence of a necrotic core in the spheroids could be controlled by changing the glucose concentration of the incubation medium. This manipulation had no effect on the size of the tumor spheroids or hypoxia in the spheroid core, which has been predicted by a mathematical model in computer simulations of spheroid growth. Control over the presence of a necrotic core while maintaining other physical parameters of the spheroid presents an opportunity to assess the impact of core necrosis on therapy efficacy. Using micro-particle imaging velocimetry (micro-PIV), we characterize the hydrodynamics and mass transport of nanoparticles in tumor spheroids in a microfluidic device. We observe a geometrical dependence on the flow rate experienced by the tumor spheroid in the device, such that the “front” of the spheroid experiences a higher flow velocity than the “back” of the spheroid. Using fluorescent nanoparticles, we demonstrate a heterogeneous accumulation of nanoparticles at the tumor interface that correlates with the observed flow velocities. The penetration depth of these nanoparticles into the tumor spheroid depends on nanoparticle diameter, consistent with reports in the literature.     

Microfluidic self-assembly of tumor spheroids for anticancer drug discovery

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of in vivo tumors. Moreover, continuous dynamic perfusion allows the establishment of physiologically relevant drug profiles to exposed spheroids. Here we present a physiologically inspired design allowing microfluidic self-assembly of spheroids, formation of uniform spheroid arrays, and characterizations of spheroid dynamics all in one platform. Our microfluidic device is based on hydrodynamic trapping of cancer cells in controlled geometries and the formation of spheroids is enhanced by maintaining compact groups of the trapped cells due to continuous perfusion. It was found that spheroid formation speed (average of 7 h) and size uniformity increased with increased flow rate (up to 10 μl min⁻¹). A large amount of tumor spheroids (7,500 spheroids per square centimeter) with a narrow size distribution (10 ± 1 cells per spheroid) can be formed in the device to provide a good platform for anticancer drug assays. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Liz Y. Wu and Dino Di Carlo contributed equally to this work.     

Three-dimensional spheroid model in tumor biology.

It is becoming more and more apparent that monolayer cultures of tumor cells cannot completely represent the characteristics of three-dimensional solid tumors. Consequently, the multicellular tumor spheroid model, which is of intermediate complexity between in vivo tumors and monolayer cultures, was developed. In this review, the major similarities between spheroids and solid tumors are discussed. After a brief survey of the different spheroid culturing techniques, the general morphological and growth characteristics of these systems are examined and compared to solid tumors. Finally, selected studies regarding the use of tumor spheroids to examine cell response to antineoplastic agents and radiation, cell death including both necrosis and apoptosis and cell adhesion in spheroids are reviewed.     

A system for viably maintaining a stirred suspension of multicellular spheroids during NMR spectroscopy

We have developed a system for the perfusion of a stirred suspension of multicellular spheroids during nuclear magnetic resonance spectroscopy. Measurement of the medium temperature, pH, oxygen tension, and glucose and lactate concentrations demonstrated that the macroenvironmental conditions around the spheroids during perfusion matched those in standard spinner culture flasks. Spheroids cultured in the NMR perfusion chamber for up to 48 h were virtually identical to spheroids cultured under standard conditions in terms of volume and cell number growth, the extent of central necrosis, cellular clonogenicity, and proliferative status. To avoid problems in interpreting the NMR spectra, we have used a medium containing 10% of the normal inorganic phosphate concentration; comparative growth and NMR studies showed that this medium had no effect on the results reported. 31P NMR spectroscopic analysis demonstrated that the mean pH, nucleotide triphosphate (NTP) to inorganic phosphate (Pi) ratio, the total amount of NTP, and the total energy charge were essentially constant over 8 h of analysis. Stopping the stirring of the spheroid culture during analysis resulted in depletion of the nucleotide phosphate pool in 30 min, with an accumulation of Pi and a shift to a more acid intracellular pH. This effect could be reversed if stirring was resumed within 30 min. Stopping the perfusion while maintaining stirring resulted in a deterioration of the 31P spectra until no high energy phosphates remained at 120 min and the pH fell to approximately 6. This effect was also partially reversible after 30 min of reperfusion, with recovery to a normal 31P spectrum requiring 10 h. The combination of the spheroid model system with 31P NMR spectroscopic analysis will provide a powerful tool for investigating basic questions about the regulation of tumor cell energy metabolism and viability.     

Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology

The growth of tumour cells as three-dimensional multicellular spheroids in vitro has led to important insights in tumour biology, since properties of the in vivo -tumour such as proliferation or nutrient gradients, can be studied under controlled conditions. While this review starts with an update of recent data on spheroid monocultures, especially concerning tumour microenvironment and therapeutic modalities, the main emphasis is put on the spectrum of heterologous cultures which have evolved in previous years. This type of culture includes tumour cell interaction with endothelial, fibroblast or immunocompetent cells. The relation of the spheroid culture model to other types of three-dimensional culture and our critical evaluation and presentation of the technical aspects of growing and analysing spheroids are included in the text. These topics are chosed to help the experimental pathologist design experiments with tumour spheroids and to stimulate discussion.     

Tethered spheroids as an in vitro hepatocyte model for drug safety screening

Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 μm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.     

Growth of primary embryo cells in a microculture system

We present optimal perfusion conditions for the growth of primary mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mESCs) using a microfluidic perfusion culture system. In an effort to balance nutrient renewal while ensuring the presence of cell secreted factors, we found that the optimal perfusion rate for culturing primary embryonic fibroblasts (mEFs) in our experimental setting is 10 nL/min with an average flow velocity 0.55 μm/s in the microchannel. Primary mEFs may have a greater dependence on cell secreted factors when compared to their immortalized counterpart 3T3 fibroblasts cultured under similar conditions. Both the seeding density and the perfusion rate are critical for the proliferation of primary cells. A week long cultivation of mEFs and mESCs using the microculture system exhibited similar morphology and viability to those grown in a petri dish. Both mEFs and mESCs were analyzed using fluorescence immunoassays to determine their proliferative status and protein expression. Our results demonstrate that a perfusion-based microculture environment is capable of supporting the highly proliferative status of pluripotent embryonic stem cells.     

The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities

Adipose-derived stem cells (ASCs) have valuable applications in regenerative medicine, but maintaining the stemness of ASCs during in vitro culture is still a challenging issue. In this study, human ASCs spontaneously formed three-dimensional spheroids on chitosan films. Most ASCs within the spheroid were viable, and the cells produced more extracellular molecules, like laminin and fibronectin. Comparing to monolayer culture, ASC spheroids also exhibited enhanced cell survival in serum deprivation condition. Although cell proliferation was inhibited in spheroids, ASCs readily migrated out and proliferated upon transferring spheroids to another adherent growth surface. Moreover, spheroid-derived ASCs exhibited higher expansion efficiency and colony-forming activity. Importantly, we demonstrated that spheroid formation of human ASCs on chitosan films induced significant upregulation of pluripotency marker genes (Sox-2, Oct-4 and Nanog). By culturing the ASC spheroids in proper induction media, we found that ASC differentiation capabilities were significantly enhanced after spheroid formation, including increased transdifferentiation efficiency into neuron and hepatocyte-like cells. In a nude mice model, we further showed a significantly higher cellular retention ratio of ASC spheroids after intramuscular injection of spheroids and dissociated ASCs. These results suggested that ASCs cultured as spheroids on chitosan films can increase their therapeutic potentials.     

Tumour fragment spheroids from human non-small-cell lung cancer maintained in organ culture

Biopsy material from 17 human non-small-cell lung carcinomas (NSCLC) was maintained in agar overlay culture as tumour fragment spheroids for 40 days. A practical procedure for the formation of spheroids and organ culture is described. The mechanically dissociated tumour specimens showed a variation in their ability to generate spheroids that was not related to the ploidy or the histological differentiation of the biopsies. Light microscopic observations revealed a heterogeneous spheroid population with a mixture of tumour cells and stromal elements. Most of the histological elements normally found in human NSCLC could be seen in the spheroids. The cellular components in the spheroids varied between highly cellular to sparsely cellular, dominated by stromal elements. The squamous carcinomas were in general found to generate highly cellular spheroids more often than the adenocarcinomas. Spheroids with a different cellular content could be selected in vitro by using a morphometric technique. Diameter measurements showed a large variability in spheroid growth. Most of the spheroids decreased in size although bromodeoxyuridine labelling indicated active cell proliferation in the specimens. Frequent changes of medium did not affect spheroid growth. The culture system presented provides a model for studying the cellular heterogeneity as well as the biological characteristics of tumour tissue from individual patients in vitro.     

振荡流动对组织工程用灌注式生物 反应器中剪切力分布影响

目的 考察振荡流动以及三维支架孔径和孔隙率对生物反应器内流速和剪切力分布的影响,并根据理论计算结果为脱细胞骨三维支架和灌注式生物反应器制备提出优化方法。方法 针对实验室前期制备的骨组织工程用脱细胞骨三维支架和灌注式生物反应器,将脱细胞骨三维支架简化为各向同性的多孔介质,对生物反应器内的流速和剪切力分布进行理论建模。结果 振荡流作用时,多孔支架材料内速度和达西剪切力呈现一致的变化规律,不同半径处流速和达西剪切力差异减小,有利于在骨组织工程中对种子细胞进行均匀三维培养。提高入口灌流速度可提高平均达西剪切力;增加多孔支架孔径或孔隙率对支架内流速峰值影响不大,但会显著降低平均达西剪切力;提高入口振荡流动振荡频率可降低支架内流速最大峰值,显著减小不同半径处流速的差异。结论 适宜的振荡流易产生利于骨组织工程干细胞所需剪切力,研究结果有望为优化骨组织工程中种子细胞的三维培养方法提供理论指导。     

Mesothelial cells interact with tumor cells for the formation of ovarian cancer multicellular spheroids in peritoneal effusions

Epithelial ovarian cancer (EOC) dissemination is primarily mediated by the shedding of tumor cells from the primary site into ascites where they form multicellular spheroids that rapidly lead to peritoneal carcinomatosis. While the clinical importance and fundamental role of multicellular spheroids in EOC is increasingly appreciated, the mechanisms that regulate their formation and dictate their cellular composition remain poorly characterized. To investigate these important questions, we characterized spheroids isolated from ascites of women with EOC. We found that in these spheroids, a core of mesothelial cells was encased in a shell of tumor cells. Analysis further revealed that EOC spheroids are dynamic structures of proliferating, non-proliferating and hypoxic regions. To recapitulate these in vivo findings, we developed a three-dimensional co-culture model of primary EOC and mesothelial cells. Our analysis indicated that, compared to the OVCAR3 cell line, primary EOC cells isolated from ascites as well as mesothelial cells formed compact spheroids. Analysis of heterotypic spheroid microarchitecture revealed a structure that grossly resembles the structure of spheroids isolated from ascites. Cells that formed compact spheroids had elevated expression of β1 integrin and low expression of E-cadherin. Addition of β1 integrin blocking antibody or siRNA-mediated downregulation of β1 integrin resulted in reduced tightness of the spheroids. Interestingly, the loss of MUC16 and E-cadherin expression resulted in the formation of more compact spheroids. Therefore, our findings support the heterotypic nature of spheroids from malignant EOC ascites. In addition, our data describe an unusual link between E-cadherin expression and less compact spheroids. Our data also emphasize the role of MUC16 and β1 integrin in EOC spheroid formation.     

Tubular perfusion system for the long-term dynamic culture of human mesenchymal stem cells

In vitro culture techniques must be improved to increase the feasibility of cell-based tissue engineering strategies. To enhance nutrient transport we have developed a novel bioreactor, the tubular perfusion system (TPS), to culture human mesenchymal stem cells (hMSCs) in three-dimensional scaffolds. This system utilizes an elegant design to create a more effective environment for cell culture. In our design, hMSCs in the TPS bioreactor are encapsulated in alginate beads that are tightly packed in a tubular growth chamber. The medium is perfused by a peristaltic pump through the growth chamber and around the tightly packed scaffolds enhancing nutrient transfer while exposing the cells to shear stress. Results demonstrate that bioreactor culture supports early osteoblastic differentiation of hMSCs as shown by alkaline phosphatase gene expression. After 14 and 28 days of culture significant increases in the gene expression levels of osteocalcin, osteopontin, and bone morphogenetic protein-2 were observed with bioreactor culture, and expression of these markers was shown to increase with media flow rate. These results demonstrate the TPS bioreactor as an effective means to culture hMSCs and provide insight to the effect of long-term shear stresses on differentiating hMSCs.     

An oxygen-permeable spheroid culture system for the prevention of central hypoxia and necrosis of spheroids

Since oxygen is one of the critical limiting factors for maintaining cell viability and function, a great deal of effort is being focused on improving the oxygen supply to three-dimensional (3D) cellular constructs. Here, we report a technique to construct spheroids utilizing 3D culture chips with a rapid and simple method for the replication of the surface structures of a polydimethylsiloxane (PDMS) mold. The resultant spheroid culture chip made it possible to rapidly yield high numbers of the spheroids at a time as well as to obtain uniform spheroids with a narrow size distribution and to collect the spheroids easily and noninvasively. The most important feature of this spheroid culture chip is that it enables direct oxygen supply to the cells because the chip is made with only gas-permeable PDMS. When human hepatoma HepG2 cells were grown on the oxygen-permeable chips as a model for liver cells, the cellular growth was remarkably enhanced, and the anaerobic glycolysis was significantly reduced compared to the non-oxygen-permeable chips. Furthermore, the oxygen-permeable chip improved the albumin secretion rates compared to the conventional spheroid culture system after 10 days. Histochemical and immunohistochemical analyses demonstrated that the oxygen-permeable chip dramatically prevented hypoxia in the core of the spheroids and subsequent central necrosis. Surprisingly, the diameters of approximately 400 and 600?μm were estimated to be the threshold of the hypoxic and survival size, respectively, for the HepG2 spheroids in the oxygenated chip. These results indicate that this chip is useful for engineering 3D cellular constructs with high viability and functionality for tissue engineering.     

Differentiation patterns in two- and three-dimensional culture systems of human squamous carcinoma cell lines.

Relative quantification of the pattern of differentiation of two squamous carcinoma cell lines of the female genital tract, A431 and CaSki, was studied in various experimental tissue culture states that are frequently used to evaluate drug and radiation effects on human tumors. Two- and three-dimensional in vitro cultures, ie, monolayers and multicellular tumor spheroids (MCTS), and nude mice-xenograft tumors as in vivo tumor models were compared. In addition, epidermal growth factor (EGF) was used comparatively in the in vitro studies. Morphologic signs of epithelial differentiation could be recognized in both cell lines gradually increasing from monolayers to MCTS to xenograft tumors. Cytokeratin (CK) expression is described as stable in A431 cells. Using immunohistochemistry, however, partial masking of CK antigens was found when applying the antibody 8.12 on monolayer cells and could be quantified by flow cytometric measurements. Fundamental cellular changes were found in a CaSki xenograft tumor, which showed newly established features of a keratinizing carcinoma after late onset of tumor growth. Epidermal growth factor caused reduction of both intercellular contacts and later onset of necrosis in MCTS, leading to an increased viability of the spheroids. Significant differences in differentiation of the tumor model systems indicates that the characterization of differentiation with immunohistochemistry and flow cytometry is necessary to assist interpretation of data obtained with these different tumor models.     

Stresses in growing soft tissues

Biochemical processes of tissue growth lead to production of new proteins, cells, and other material particles at the microscopic level. At the macroscopic level, growth is marked by the change of the tissue shape and mass. In addition, the appearance of the new material particles is generally accompanied by deformation and, consequently, stresses in the surrounding material. Built upon a microscopic toy-tissue model mimicking the mechanical processes of mass supply, a simple phenomenological theory of tissue growth is used in the present work for explaining residual stresses in arteries and studying stresses around growing solid tumors/multicell spheroids. It is shown, in particular, that the uniform volumetric growth can lead to accumulation of residual stresses in arteries because of the material anisotropy. This can be a complementary source of residual stresses in arteries as compared to the stresses induced by non-uniform tissue growth. It is argued that the quantitative assessment of the residual stresses based on in vitro experiments may not be reliable because of the essential stress redistribution in the tissue samples under the cutting process. Concerning the problem of tumor growth, it is shown that the multicell spheroid or tumor evolution depends on elastic properties of surrounding tissues. In good qualitative agreement with the experimental in vitro observations on growing multicell spheroids, numerical simulations confirm that stiff hosting tissues can inhibit tumor growth.     

Localization of CD44 at the Invasive Margin of Glioblastomas by Immunoelectron Microscopy

Glioblastoma multiforme is a highly invasive primary brain tumor, which is known to strongly express the CD44 cell adhesion receptor. A number of experimental studies suggest that the interaction of this receptor with extracellular matrix (ECM) proteins such as hyaluronic acid may in part mediate human glioma cell adhesion and invasion of brain tissue. Although the expression of CD44 and its spliced variants in brain tumors have been extensively studied, there have been no reports localizing its expression to the invasive margin of the tumor. The authors used immunoelectron microscopy to investigate the expression patterns of CD44 in an in vitro organotypic invasion assay. Tumor spheroids initiated from the U373 MG human glioblastoma line were confronted with fetal rat brain aggregates in a spheroid coculture system. The CD44 expression appeared at the interface between glioblastoma tumor spheroids and brain tissue, as well as in the spheroid itself. CD44 immunoreactivity was not detectable in mature 21-day fetal brain aggregates. The findings provide direct evidence that CD44 is expressed at the confrontational invasive border between glioblastomas and brain tissue, further supporting its role in glioma cell-ECM recognition and attachment.     

Periodic “flow-stop” perfusion microchannel bioreactors for mammalian and human embryonic stem cell long-term culture

The present study examines the use of automated periodic “flow-stop” perfusion systems for long-term culture of mammalian cells in a microchannel bioreactor. The method is used to culture Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) for long periods of time (>7 d) in a microchannel (height 100 μm). Design parameters, mass transport and shear stress issues are theoretically examined via numerical simulations. Cell growth and morphology are experimentally monitored and an enhanced growth rate was measured compared to constant perfusion micro-reactors and to traditional culture in Petri dishes. Moreover, we demonstrate the use of the method to co-culture undifferentiated colonies of human Embryonic Stem Cells (hESC) on HFF feeder cells in microchannels. The successful hESC-HFF co-culture in the microbioreactor is achieved due to two vital characteristics of the developed method—short temporal exposure to flow followed by long static incubation periods. The short pulsed exposure to shear enables shear sensitive cells (e.g., hESC) to withstand the medium renewal flow. The long static incubation period may enable secreted factors (e.g., feeder cells secreted factors) to accumulate locally. Thus the developed method may be suitable for long-term culture of sensitive multi-cellular complexes in microsystems.     

The effects of spheroid formation of adipose-derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential

Adipose-derived stem cells (ADSCs) represent a valuable source of stem cells for regenerative medicine, but the loss of their stemness during in vitro expansion remains a major roadblock. We employed a microgravity bioreactor (MB) to develop a method for biomaterial-free-mediated spheroid formation to maintain the stemness properties of ADSCs. ADSCs spontaneously formed three-dimensional spheroids in the MB. Compared with monolayer culture, the expression levels of E-cadherin and pluripotent markers were significantly upregulated in ADSC spheroids. Spheroid-derived ADSCs exhibited increased proliferative ability and colony-forming efficiency. By culturing the spheroid-derived ADSCs in an appropriate induction medium, we found that the multipotency differentiation capacities of ADSCs were significantly improved by spheroid culture in the MB. Furthermore, when ADSCs were administered to mice with carbon tetrachloride-induced acute liver failure, spheroid-derived ADSCs showed more effective potentials to rescue liver failure than ADSCs derived from constant monolayer culture. Our results suggest that spheroid formation of ADSCs in an MB enhances their stemness properties and increases their therapeutic potential. Therefore, spheroid culture in an MB can be an efficient method to maintain stemness properties, without the involvement of any biomaterials for clinical applications of in vitro cultured ADSCs.     

Using Cartilage to Repair Bone: An Alternative Approach in Tissue Engineering

Materials and techniques currently used for bone replacement/repair conform to the current paradigm, relying on bone or bone products to produce bone or induce bone formation. Yet, nature forms and heals most of the skeleton by ossification of a cartilaginous model. In this study, we cultured aggregates of E10.5 or E12 mouse embryonic limb cells in the bioreactor for 3 weeks, determined the stages of cartilage differentiation attained, and assessed the ossification and bone healing potential of the spheroids by implantation adjacent to, or directly in, a skull defect. Cultured spheroids had large cartilaginous areas, sometimes with cellular arrangements characteristic of growth plate zones. Aggregates implanted for 2 weeks adjacent to a defect mineralized and ossified (histology, micro-CT). Defects with implants had a central mass of differentiated and differentiating bone, with osteoclast activity, filling the defect. Controls had considerable remodeling on the bone edges demarcating the still present defect. This study shows that cartilage, grown in the bioreactor for 3 weeks, ossified when implanted adjacent to a bone defect, and when implanted directly in a defect, contributed to its healing. Our ability to grow differentiated bone-forming cartilage for implantation is an alternative approach in the field of bone repair.