Cell Culture Parameters Research Articles

Challenging Small Human Hepatocytes with Opiates: Further Characterization of a Novel Prototype Bioartificial Liver

Bioartificial liver (BAL) systems can take over liver functions in patients undergoing liver failure until transplantation. Recently, a novel prototype rotary BAL has been developed using small human hepatocytes (SH). This study investigated the metabolism of opiates morphine and methadone in the BAL and their influence on the basic cell culture parameters, viability, and growth of SH. Opiates may be present in patients due to pain therapy, anticancer treatment, or drug abuse. Cells were cultivated in the BAL for a total of 12 days and exposed twice to 100 microg/L of morphine or methadone. Morphine and methadone concentrations were analyzed using gas chromatography with a mass spectrometry detector. Further, the production of albumin, lactate dehydrogenase release, lactate release, urea production, and glucose consumption were measured. Cell viability and growth were determined by confocal microscopy. Cytochrome P 3A4 and uridindiphosphat (UDP) glucuronosyl transferase 2B7 in SH were analyzed by western blot. The mean cell density during treatment was 5.5 +/- 0.7 x 10(6) cells/mL (n = 6) and was not altered significantly by the opiates. Cell viability stayed above 90%. Morphine was not reduced by SH and was a stress factor as determined by decreased metabolic activity. On the other hand, SH metabolized methadone showing first-order kinetics: the first-order rate constant k = 0,019, half-life t(1/2) = 36 h. Methadone metabolism led to decreased urea and albumin production. The expression of cytochrome P 3A4, mainly responsible for methadone metabolism, was proved in SH. The prototype BAL is basically suited to support liver functions, provided patients receive therapy with methadone.

In Vitro Biocompatibility of Sheath–Core Cellulose-Acetate-Based Electrospun Scaffolds Towards Endothelial Cells and Platelets

Typically, tissue-engineered scaffolds mimic the topographical properties of the native extracellular matrix. However, other physical properties, such as the scaffold mechanical stiffness, are not imitated. The purpose of this study was to fabricate scaffolds with improved mechanical properties and investigate their biocompatibility towards endothelial cells and platelets. To enhance mechanical properties, an electrospinning apparatus was developed that fabricates fibers with sheath–core morphologies. Different combinations of cellulose acetate and chitosan were chosen to modulate the mechanical properties of the formed fibers. We hypothesized that mechanically stiffer scaffolds would improve endothelial cell growth and that all scaffolds would be compatible towards endothelial cells and platelets. Endothelial cell-culture conditions were quantified up to 5 days. Migration onto scaffolds was monitored for 10 days. Platelet aggregation, antagonized by thrombin receptor agonist peptide 6, was measured after scaffold incubation. A platelet activation time-course was assessed with the modified prothrombinase assay. As scaffold mechanical stiffness increased, endothelial cell growth within and adhesion to and migration throughout the scaffolds was promoted. Also, scaffolds did not induce platelet aggregation or activation. These results indicate that the mechanical stiffness of engineered scaffolds regulates endothelial cell-culture parameters and that these sheath–core electrospun scaffolds are compatible towards endothelial cells and platelets.

Degradation of an Fc‐fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease

A host-cell-related proteolytic activity was identified in a recombinant Fc-fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post-capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo-protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters.

Influence of culture aeration on the cellulase activity of Thermobifida fusca

Currently, one of the hurdles hindering efficient production of cellulosic biofuel is the recalcitrant nature of cellulose to hydrolysis. A wide variety of cellulase enzymes are found natively in microorganisms that can potentially be used to effectively hydrolyze cellulose to fermentable sugars. In this study, phenomenological and mechanistic parameters affecting cellulase activity were studied using the moderately thermophilic, aerobic, and cellulolytic microorganism Thermobifida fusca. Two major sets of experiments were conducted to (1) study the mechanistic differences in growth in a flask compared to a bioreactor and (2) study the cell culture parameters influencing cellulase activity using a series of bioreactor experiments. Specific cellulase and specific endoglucanase activities were found to be higher in the bioreactor as compared to flask growth. Measurements of messenger RNA transcript levels of 18 cellulase-related genes and intracellular ATP levels indicated that measured enzyme activity was likely more influenced by post-transcriptional energetics rather than transcriptional regulation. By delineating the effects of culture aeration and stir speed using a bioreactor, it was found that cellulase activity increased with increasing aeration and increasing stir speeds (highest K(l)a) with a tradeoff of decreased cellular growth at the highest stir speeds tested (400 rpm). Overall, these results allude to a connection between aeration and oxidative respiration that lead to increased ATP allowing for increased cellulase synthesis as the primary constraint on overall cellulase activity.

Computational tools for assessing the properties of 2D neural cell cultures

Neurons cultured in vitro provide a particularly promising experimental system for the analysis of properties, such as information coding, transmission, and learning, that are conventionally associated with biological neural networks. In these systems, isolated cells are placed on top of a recording plate (microelectrode array, MEA), where they spontaneously develop a random connectivity structure. Typical cultures consist of several thousands of neurons and the connectivity density varies from very low at the beginning of an experimental trial to high in mature cultures. In the absence of external stimuli, a culture exhibits a typical pattern of spontaneous activity, alternating intervals of slow spiking and bursting with the transition intervals of increasing activity. Spontaneous activity recorded in the cultures of rat cortical cells is described in [1,2] and an explanation of the phenomena is proposed in [3]. The behavior in the presence of external stimuli is also reported in the literature, for example, the adaptation exhibited in the presence of frequent and rare stimuli is assessed experimentally and through a computational model in [4]. The present work is related to the previously reported study [3] in which an image-processing algorithm is used to detect some structural parameters of cell cultures. A typical result from this study is illustrated in Figure 1. The original image of cultured cells on top of recording plate is shown in panel A, one of its segments in B, and the result of the applied algorithm in C. The blue pattern on panel C corresponds to cells. This approach, in general, enables automated estimation of parameters like the number of cells, or the average density of connections between the cells. Here, we propose a computational model based on the study in [3]. The neural network model is composed of leaky integrate-and-fire neurons, connected in a recurrent network as shown in panel D. The network is fed with the quantitative information about the structure of the cell cultures. Such model, although approximate, captures well the essential properties of the topologies observed in cultures. The presented model is used to reproduce and analyze network behavior observed in the absence of external stimuli. The structural parameters are estimated in different phases of development to closely relate them to the observed behavior. The relation between the network topology and behavior is systematically examined throughout this study.

Cartilage repair: past and future – lessons for regenerative medicine

Since the first cell therapeutic study to repair articular cartilage defects in the knee in 1994, several clinical studies have been reported. An overview of the results of clinical studies did not conclusively show improvement over conventional methods, mainly because few studies reach level I of evidence for effects on middle or long term. However, these explorative trials have provided valuable information about study design, mechanisms of repair and clinical outcome and have revealed that much is still unknown and further improvements are required. Furthermore, cellular and molecular studies using new technologies such as cell tracking, gene arrays and proteomics have provided more insight in the cell biology and mechanisms of joint surface regeneration. Besides articular cartilage, cartilage of other anatomical locations as well as progenitor cells are now considered as alternative cell sources. Growth Factor research has revealed some information on optimal conditions to support cartilage repair. Thus, there is hope for improvement. In order to obtain more robust and reproducible results, more detailed information is needed on many aspects including the fate of the cells, choice of cell type and culture parameters. As for the clinical aspects, it becomes clear that careful selection of patient groups is an important input parameter that should be optimized for each application. In addition, the study outcome parameters should be improved. Although reduced pain and improved function are, from the patient's perspective, the most important outcomes, there is a need for more structure/tissue-related outcome measures. Ideally, criteria and/or markers to identify patients at risk and responders to treatment are the ultimate goal for these more sophisticated regenerative approaches in joint surface repair in particular, and regenerative medicine in general.

Biocompatibility of surfactant‐templated polyurea–nanoencapsulated macroporous silica aerogels with plasma platelets and endothelial cells

The recently synthesized polyurea-nanoencapsulated surfactant-templated aerogels (X-aerogels) are porous materials with significantly improved mechanical strengths. Surface-wise they resemble polyurethane, a common biocompatible material, but their biocompatibility has never been investigated. As lightweight and strong materials, if X-aerogels also have acceptable biocompatibility, they may be used in many implantable devices. The goal of this study was to investigate their biocompatibility toward platelets, blood plasma, and vascular endothelial cells, in terms of cell activation and inflammatory responses. Platelets were incubated with X-aerogel and platelet activation was measured through CD62P and phosphatidylserine expression. Platelet aggregation was also measured. Contact with X-aerogel did not induce platelet activation or impair aggregation. To determine X-aerogel-induced inflammation, plasma anaphylatoxin C3a level was measured after incubation with X-aerogel. Results showed that X-aerogel induced no changes in plasma C3a levels. SEM and SDS-PAGE were used to examine cellular/protein deposition on X-aerogel samples after plasma incubation. No structural change or organic deposition was detected. Furthermore, X-aerogel samples did not induce any significant changes in vascular endothelial cell culture parameters after 5 days of incubation. These observations suggest that X-aerogels have a suitable biocompatibility toward platelets, plasma, and vascular endothelial cells, and they have potential for use in blood implantable devices.

Optimization of primary culture condition for mesenchymal stem cells derived from umbilical cord blood with factorial design

Mesenchymal stem cells (MSCs) can not only support the expansion of hematopoietic stem cells in vitro, but also alleviate complications and accelerate recovery of hematopoiesis during hematopoietic stem cell transplantation. However, it proved challenging to culture MSCs from umbilical cord blood (UCB) with a success rate of 20-30%. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In this work, fractional factorial design was applied to study the effect of cell inoculated density, combination and dose of cytokines, and presence of serum and stromal cells. The cultured UCB-MSC-like cells were characterized by flow cytometry and their multilineage differentiation potentials were tested. The optimal protocol was identified achieving above 90% successful outcome: 2 x 10(6) cells/mL mononuclear cells inoculated in Iscove's modified Dulbecco's medium supplied with 10% FBS, 15 ng/mL IL-3, and 5 ng/mL Granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, the UCB-MSC-like cells expressed MSC surface markers of CD13, CD29, CD105, CD166, and CD44 positively, and CD34, CD45, and human leukocyte antigens-DR (HLA-DR) negatively. Meanwhile, these cells could differentiate into osteoblasts, chondrocytes, and adipocytes similarly to MSCs derived from bone marrow. In conclusion, we have developed an efficient protocol for the primary culture of UCB-MSCs by adding suitable cytokines into the culture system.

The effects of cell culture parameters on cell release kinetics from thermoresponsive surfaces

The thermoresponsive properties of poly(N-isopropyl acrylamide) (pNIPAM) have led to its wide use in bioengineering applications, including the reversible adhesion of mammalian cells. The groups performing this research have used different solutions to initiate cell release and have varied the temperature of the solution during detachment. To our knowledge, there has been no direct correlation between the solution identity or temperature on the efficiency of cell release from pNIPAM films. In this work, we present a study of the effect of the solution type and temperature used to initiate detachment on the time required to achieve 100% detachment of bovine aortic endothelial cells (BAECs) from pNIPAM. The pNIPAM films used in this work were obtained using a novel technique using a spin-coated solution containing pNIPAM (spNIPAM). We found that the fastest, most reliable release of cells occurred below the LCST of the polymer at 4 degrees C in serum free media (SFM). As it is sometimes desirable to stop cell metabolism at the time of detachment (e.g., to "freeze" protein expression prior to subsequent analysis), the use of extremely cold SFM would be ideal in such cases.

Effect of Pluronic F-68, 5% CO, Atmosphere, HEPES, and Antibiotic-Antimycotic on Suspension Adapted 293 Cells

The influence of different parameters upon cell culture of serum-free adapted 293 cells including the surfactant Pluronic F-68, carbon dioxide (5% CO2 atmosphere), buffer HEPES and antibiotic-antimycotic has been explored. A defined serum-free medium (SFM) formulated without human or animal origin components from Invitrogen was used to grow the suspension adapted 293 cells. For all cell culture parameters cell density and viability of the suspension adapted 293 cells were monitored. The results indicated that the PF68 concentrations ranging from 0.05% to 0.2% can be used in the culture of the suspension adapted 293 cells since no negative effect upon either cell density or viability was detected. This will minimize the formation of aggregates during cell culture. It was demonstrated that neither the cell density nor the viability of the suspension adapted 293 cells were affected by the 5% CO2 atmosphere at the inoculation cell densities evaluated. The use of the buffer HEPES in the cultivation of suspension adapted 293 cells did not cause negative effects upon cell density and viability. The addition of HEPES makes more robust the culture to pH fluctuations. The antibioticantimycotic can be used when needed at concentrations of up to 50 IU/ml for the culture of this particular cell line, with no apparent effect upon cell growth. The results obtained will contribute to a basic understanding of the 293 cell culture in the 293 SFM II and to the process development of their culture in bioreactors for the expression of different products of biotechnology interest.

Production of Adenoviral Vectors in 293 Cells: A Case Study of the Adaptation of Attached Cells to Grow in Suspension

A study of the production of adenoviral vectors in suspension 293 cells has been explored. A defined serumfree medium (293 SFM II) formulated without human or animal origin components from Invitrogen was used for the suspension adapted 293 cells. It was demonstrated that the 293 cells can be adapted to grow in suspension using serum free medium. The effect of different cell culture parameters was determined. The production technique demonstrated here is expected to simplify purification processes and circumvents the problems associated with serum containing medium.

Cytotoxicity of resin composites as a function of interface area

Objectives The standardization protocols for biomaterial cytotoxicity testing require fine tuning for oral biomaterials to obtain international comparability as the basis for risk assessment. The principal aims were specifically to evaluate the effect of (i) relative interface area (ratio of specimen surface to cell layer surface) and (ii) volume of cell culture medium on cytotoxicity as a potential modification of ISO 10993-5. Methods ISO 10993-5 was followed with an interface area of 12.5%, as recommended, using primary human gingival fibroblasts and L-929 mouse fibroblasts. In another series of experiments (using L-929 cells) the interface area was varied between 12.5% and 0.71%. For each relative interface area, three conditions for affecting the cure of the resin composite were investigated by using three mould materials: white, transparent and black moulds. In addition, the volume of cell culture medium was varied. Composite specimens (Herculite XRV) were added to the cultures immediately after production or preincubation for 1, 2, 7 days or 6 weeks under cell culture conditions. Specimens were incubated with fibroblasts for 72 h and cell numbers determined by flow cytometry. Glass specimens resembling composite specimens in diameter and height were used as negative controls. Results Cytotoxicity results with primary gingival fibroblasts were comparable to results with the cell line L-929. An effect from the color/material of the specimen moulds was found. Different ratios of specimen sizes to cell culture parameters (cell layer surface, volume of cell culture medium) produced different results. Three out of four differently designed specimens showed the same behavior in cell culture. Significance Cytotoxicity tests should be further standardized in line with existing standards with regard to specimen production protocols to ensure results are internationally comparable to validate these tests as tools for risk assessment.

Electrical stimulation enhances viability of human cutaneous fibroblasts on conductive biodegradable substrates

Electrically conductive biodegradable polymer membranes were prepared by mixing conductive polypyrrole particles with poly(L-lactic acid) solution followed by solution casting and solvent evaporation. Multi-well electrical cell culture plates were fabricated to host electrically stimulated cell culture and monitor parameters. Human cutaneous fibroblasts were cultured on conductive membranes with or without electrical stimulation (ES). Cell count, MTT, Hoechst staining, and SEM were performed to characterize the cells. The membranes supported the adhesion and proliferation of the fibroblasts in both the presence and absence of ES. In the presence of direct electrical field strength of 100 mV/mm, cell viability on the PPy/PLLA membranes at 2 and 24 h was 2.2- and 4.0-fold (p < 0.05) respectively of that on the same membranes without ES. Direct electrical current ranging from 2.5 to 250 microA/mm had no effect on the viability of cells cultured on the gold-coated Petri dish. Electrical field applied to conductive biodegradable polymer surfaces is therefore an effective approach to upregulate the mitochondrial activity of human skin fibroblasts.

Head and neck squamous cell carcinoma cell lines: Established models and rationale for selection

Head and neck squamous cell carcinoma (HNSCC) cell lines are important preclinical models in the search for novel and targeted therapies to treat head and neck cancer. Unlike many other cancer types, a wide variety of primary and metastatic HNSCC cell lines are available. An easily accessible guide that organizes important characteristics of HNSCC cell lines would be valuable for the selection of appropriate HNSCC cell lines for in vitro or in vivo studies. A literature search was performed. Cell growth and culture parameters from HNSCC cell lines were catalogued into tables or lists of selected characteristics. Methods for establishing cancer cell lines and basic cell culture maintenance techniques were reviewed. A compendium of HNSCC cell line characteristics is useful for organizing the accumulating information regarding cell line characteristics to assist investigators with the development of appropriate preclinical models.

Considerations on the lactate consumption by CHO cells in the presence of galactose

A CHO cell line producing t-PA was cultured using glutamate and glucose or galactose to decrease the formation of metabolic end-products and therefore improving the process. In batch cultures using glutamate (6 mM) with glucose at two different levels (5 and 20 mM) or with glucose and galactose (5 and 20 mM, respectively) a remarkable difference in cell culture parameters was evidenced. For 20 mM glucose, a usual cell pattern was observed with lactate built-up in the medium. For 5 mM glucose, cell growth was arrested due to glucose depletion and only a limited use of the excreted lactate could be observed, not supporting cell growth sufficiently. However, when glucose 5 mM and galactose 20 mM were used together, cells consumed the glucose first and, interestingly, in a second phase they continued growing on galactose with the simultaneous consumption of the endogenous lactate. Under these conditions, cell growth was even improved with respect to growth on 20 mM glucose, used as a control. This metabolic behavior is further investigated by using metabolic flux analysis, suggesting that the lactate produced is not used in the oxidative metabolism through the TCA cycle. Metabolic fate of the lactate consumed is discussed.

Alginate‐PLL microencapsulation: Effect on the differentiation of embryonic stem cells into hepatocytes

The emergence of hepatocyte based clinical and pharmaceutical technologies, has been limited by the absence of a stable hepatocyte cell source. Embryonic stem cells may represent a potential solution to this cell source limitation problem since they are highly proliferative, renewable, and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, their practicality is limited by: (1) low yields of fully differentiated cells, (2) absence of large scale processing considerations, and (3) ineffective downstream enrichment protocols. Thus, a differentiation platform that may be modified to induce and sustain differentiated cell function and scaled to increase differentiated cell yield would improve current stem cell differentiation strategies. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as the diffusion of growth factors, metabolites, and wastes. In addition, both cell seeding density and bead composition may be manipulated. In order to assess the feasibility of directing stem cell differentiation via microenvironment regulation, we have developed a murine embryonic stem cell (ES) alginate poly-l-lysine microencapsulation hepatocyte differentiation system. Our results indicate that the alginate microenvironment maintains cell viability, is conducive to ES cell differentiation, and maintains differentiated cellular function. This system may ultimately assist in developing scalable stem cell differentiation strategies.

Posttransplant hematopoiesis in patients undergoing sibling allogeneic stem cell transplantation reflects that of their respective donors although with a lower functional capability

We tested the principle of whether patient long-term hematopoiesis following allogeneic stem cell transplantation (allo-SCT) reflects the characteristics of the hematopoiesis of their respective donor. For this purpose, we analyzed bone marrow (BM) hematopoiesis using long-term cultures (LTC), delta assays, and clonogeneic assays as well as CD34+ cells and their subsets by flow cytometry in a series of 37 patients undergoing allo-SCT, and we compared it to that of their respective human leukocyte antigen-matched sibling donors in a paired study performed more than 1 year after the transplant procedure. Interestingly, the main factor that influenced post-allo-SCT BM hematopoiesis in the long term was donor hematopoiesis. Nevertheless, compared to their respective donors, patients exhibited a significantly lower number of colony-forming units granulomonocytic, burst-forming units erythroid, and immature progenitors (CD34++/CD38dim/CD90+/CD133+ cells, LTC-initiating cells, and colonies generated in the delta assay). Moreover, BM stromal function was diminished in patients undergoing allo-SCT compared to their donors. In addition, the presence of chronic graft-versus-host disease under immunosuppressive treatment also conditioned an impaired hematopoietic function. In summary, our study shows that BM hematopoiesis evaluated more than 1 year after an allo-SCT mainly reproduces that of their respective donors, although with a significantly decreased in vitro activity.

Improved Structure−Activity Relationship Analysis of HIV-1 Protease Inhibitors Using Interaction Kinetic Data

Despite the availability of large amounts of data for HIV-protease inhibitors and their effectiveness with wild type and resistant enzyme, there is limited knowledge about how this and other information can be systematically applied to the development of new antiviral compounds. To identify in vitro parameters that correlate with the efficacy of HIV inhibitors in cell culture, the relationships between inhibition, interaction kinetic, and cell culture parameters for HIV-1 protease inhibitors were analyzed. Correlation, cluster, and principal component analysis of data for 37 cyclic and linear compounds revealed that the affinities (K(D)) determined from SPR-biosensor binding studies correlated better to cell culture efficacy (ED(50)) than that of the inhibition constants (K(i)), indicating that the conventional use of K(i) values for structure-activity relationship analysis of HIV-1 inhibitors should be seriously reconsidered. The association and dissociation kinetic rate constants (k(on) and k(off)) alone showed weak correlations with ED(50) values. However, ED(50) values were most related to the free enzyme concentration in the viral particle ([E]), calculated from the rate constants and the total enzyme concentration in a viral particle. A structure-activity relationship analysis of the current data set was found to be valid for all classes of compounds analyzed. In summary, use of affinity, based on interaction kinetic rate constants, rather than inhibition constants, and theoretical consideration of the physiological conditions in the virus particle provide improved structure-activity relationship analysis of HIV-1 protease inhibitors.

24-Epibrassinolide at Subnanomolar Concentrations Modulates Growth and Production Characteristics of a Mouse Hybridoma

Brassinosteroids are known to stimulate plant growth and to possess antistress activities in plants. This work was aimed at exploring possible beneficial effects of 24-epibrassinolide on cultured mammalian cells. A mouse hybridoma was cultured either in standard serum-free medium, or in medium diluted to 30%, in which the cells underwent nutritional stress. Steady-state parameters of semicontinuous cultures conducted at 24-epibrassinolide concentrations from 10-16 to 10-9 mol l-1 were evaluated. Typical effects of the agent found both in standard and in diluted media were (i) increase in the value of mitochondrial membrane potential, (ii), drop of intracellular antibody level, (iii) increase in the fraction of the cells in the G0/G1 phase, and (iv) decrease in the fraction of the cells in the S phase. Alleviation of nutritional stress manifested itself in cultures conducted in diluted media. Viable cell density was significantly higher (relative to control) at 24-epibrassinolide concentrations 10-13 and 10-12 mol l-1. The results of this exploratory study show that the plant hormone 24-epibrassinolide may induce perturbations in the cell division mechanism, in mitochondria performance, and in secreted protein synthesis in a mammalian cell line. At the lowest brassinosteroid concentrations, the number of steroid molecules in the culture was of the same order of magnitude as the number of viable cells in the culture. This implies involvement of a complex cascade mechanism, through which the steroid molecule induces alterations in gene expression leading finally to significant changes in cell culture parameters.

Mitochondrial activity in response to serum starvation in bovine (Bos taurus) cell culture.

In nuclear transfer procedures, in addition to nuclei, donor cell mitochondria are routinely transferred into recipient oocytes, and mitochondrial heteroplasmy has been reported. However, various protocols have resulted in either homoplasmy for recipient oocyte mitochondria or varying heteroplasmic levels in cloned animals. In nuclear transfer protocols, donor cells are subjected to serum-starvation prior to electroporation. Therefore, the relationship between culture conditions and mitochondrial activity was explored. Fibroblast cell lines were propagated from bovine ear epithelium, skin, skeletal muscle, or cumulus cells. In vitro mitochondrial viability was assessed in proliferative and confluent cells, cultured under serum-starvation or supplemented conditions. Cells were stained with MitoTracker Red CMXRos and comparative fluorescence intensities were assessed. The mitochondrial activity per cell was highest under proliferation, significantly lower at confluency (p < 0.001), and remained depressed after serum starvation for within a week (p < 0.001). Serum starvation induced an increase in mitochondrial viability in confluent cells. These results demonstrate that mitochondrial viability is dramatically affected by cell culture conditions. Consequently, specific cell culture parameters provide one explanation for the varying incidence of heteroplasmy identified in cloned animals. Future research should reveal whether specific cell culture parameters represent one of the factors for the varying incidence of heteroplasmy identified in cloned animals.