Evaluation of p24-based antiretroviral treatment monitoring in pediatric HIV-1 infection: prediction of the CD4+ T-cell changes between consecutive visits

Worldwide, 700,000 infants are infected annually by HIV-1, most of them in resource-limited settings. Care for these children requires simple, inexpensive tests. We have evaluated HIV-1 p24 antigen for antiretroviral treatment (ART) monitoring in children. p24 by boosted enzyme-linked immunosorbent assay of heated plasma and HIV-1 RNA were measured prospectively in 24 HIV-1-infected children receiving ART. p24 and HIV-1 RNA concentrations and their changes between consecutive visits were related to the respective CD4+ changes. Age at study entry was 7.6 years; follow-up was 47.2 months, yielding 18 visits at an interval of 2.8 months (medians). There were 399 complete visit data sets and 375 interval data sets. Controlling for variation between individuals, there was a positive relationship between concentrations of HIV-1 RNA and p24 (P < 0.0001). While controlling for initial CD4+ count, age, sex, days since start of ART, and days between visits, the relative change in CD4+ count between 2 successive visits was negatively related to the corresponding relative change in HIV-1 RNA (P = 0.009), but not to the initial HIV-1 RNA concentration (P = 0.94). Similarly, we found a negative relationship with the relative change in p24 over the interval (P < 0.0001), whereas the initial p24 concentration showed a trend (P = 0.08). Statistical support for the p24 model and the HIV-1 RNA model was similar. p24 may be an accurate low-cost alternative to monitor ART in pediatric HIV-1 infection.

Differential response of porcine osteoblasts and chondrocytes in cell or tissue culture after 5-aminolevulinic acid-based photodynamic therapy

OBJECTIVE: Outcome in osteochondral allografting is limited by the immunological incompatibility of the grafted tissue. Based on a resistance of chondrocytes to photodynamic therapy in cell culture it is proposed that 5-aminolevulinic acid-based photodynamic therapy (5-ALA-PDT) might be used to inactivate bone while maintaining viability of chondrocytes and thus immunomodulate bone selectively. METHODS: Chondrocytes and osteoblasts from porcine humeral heads were either isolated (cell culture) or treated in situ (tissue culture). To quantify cytotoxic effects of 5-ALA-PDT (0-20J/cm(2), 100mW/cm(2)) an (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide) (MTT)-assay was used in cell culture and in situ hybridization in tissue culture to assess metabolic active cells (functional osteoblasts: colalpha(1)(I) mRNA, functional chondrocytes: colalpha(1)(II) mRNA). RESULTS: In cell culture, survival after 5-ALA-PDT was significantly higher for chondrocytes (5J/cm(2): 87+/-12% compared to untreated cells) than for osteoblasts (5J/cm(2): 12+/-11%). In tissue culture, the percentage of functional chondrocytes in cartilage showed a decrease after 5-ALA-PDT (direct fixation: 92+/-2%, 20J/cm(2): 35+/-15%; P<0.0001). A significant decrease in the percentage of bone surfaces covered by functional osteoblasts was observed in freshly harvested (31+/-3%) compared to untreated tissues maintained in culture (11+/-4%, P<0.0001), with no further decrease after 5-ALA-PDT. CONCLUSION: Chondrocytes were more resistant to 5-ALA-PDT than osteoblasts in cell culture, while in tissue culture a loss of functional chondrocytes was observed after 5-ALA-PDT. Since osteoblasts - but not chondrocytes - were sensitive to the tissue culture conditions, devitalized bone with functional cartilage might already be achieved by applying specific tissue culture conditions even without 5-ALA-PDT.

Clinical significance of cell cycle- and apoptosis-related markers in biliary tract cancer: a tissue microarray-based approach revealing a distinctive immunophenotype for intrahepatic and extrahepatic cholangiocarcinomas

Cholangiocarcinoma is the second most common malignant tumor of the liver. We analyzed, immunohistochemically, the significance of cell cycle- and apoptosis-related markers in 128 cholangiocarcinomas (42 intrahepatic, 70 extrahepatic, and 16 gallbladder carcinomas) combined in a tissue microarray. Follow-up was available for 57 patients (44.5%). In comparison with normal tissue (29 specimens), cholangiocarcinomas expressed significantly more frequently p53, bcl-2, bax, and COX-2 (P.05 <). Intrahepatic tumors were significantly more frequently bcl-2+ and p16+, whereas extrahepatic tumors were more often p53+ (P < .05). Loss of p16 expression was associated with reduced survival of patients. Our data show that p53, bcl-2, bax, and COX-2 have an important role in the pathogenesis of cholangiocarcinomas. The differential expression of p16, bcl-2, and p53 between intrahepatic and extrahepatic tumors demonstrates that there are location-related differences in the phenotype and the genetic profiles of these tumors. Moreover, p16 was identified as an important prognostic marker in cholangiocarcinomas.

Characterization of thermal and mechanical properties of polypropylene-based composites for fuel cell bipolar plates and development of educational tools in hydrogen and fuel cell technologies

In this project we developed conductive thermoplastic resins by adding varying amounts of three different carbon fillers: carbon black (CB), synthetic graphite (SG) and multi-walled carbon nanotubes (CNT) to a polypropylene matrix for application as fuel cell bipolar plates. This component of fuel cells provides mechanical support to the stack, circulates the gases that participate in the electrochemical reaction within the fuel cell and allows for removal of the excess heat from the system. The materials fabricated in this work were tested to determine their mechanical and thermal properties. These materials were produced by adding varying amounts of single carbon fillers to a polypropylene matrix (2.5 to 15 wt.% Ketjenblack EC-600 JD carbon black, 10 to 80 wt.% Asbury Carbon's Thermocarb TC-300 synthetic graphite, and 2.5 to 15 wt.% of Hyperion Catalysis International's FIBRILTM multi-walled carbon nanotubes) In addition, composite materials containing combinations of these three fillers were produced. The thermal conductivity results showed an increase in both through-plane and in-plane thermal conductivities, with the largest increase observed for synthetic graphite. The Department of Energy (DOE) had previously set a thermal conductivity goal of 20 W/m·K, which was surpassed by formulations containing 75 wt.% and 80 wt.% SG, yielding in-plane thermal conductivity values of 24.4 W/m·K and 33.6 W/m·K, respectively. In addition, composites containing 2.5 wt.% CB, 65 wt.% SG, and 6 wt.% CNT in PP had an in–plane thermal conductivity of 37 W/m·K. Flexural and tensile tests were conducted. All composite formulations exceeded the flexural strength target of 25 MPa set by DOE. The tensile and flexural modulus of the composites increased with higher concentration of carbon fillers. Carbon black and synthetic graphite caused a decrease in the tensile and flexural strengths of the composites. However, carbon nanotubes increased the composite tensile and flexural strengths. Mathematical models were applied to estimate through-plane and in-plane thermal conductivities of single and multiple filler formulations, and tensile modulus of single-filler formulations. For thermal conductivity, Nielsen's model yielded accurate thermal conductivity values when compared to experimental results obtained through the Flash method. For prediction of tensile modulus Nielsen's model yielded the smallest error between the predicted and experimental values. The second part of this project consisted of the development of a curriculum in Fuel Cell and Hydrogen Technologies to address different educational barriers identified by the Department of Energy. By the creation of new courses and enterprise programs in the areas of fuel cells and the use of hydrogen as an energy carrier, we introduced engineering students to the new technologies, policies and challenges present with this alternative energy. Feedback provided by students participating in these courses and enterprise programs indicate positive acceptance of the different educational tools. Results obtained from a survey applied to students after participating in these courses showed an increase in the knowledge and awareness of energy fundamentals, which indicates the modules developed in this project are effective in introducing students to alternative energy sources.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Prediction of total quarter milk somatic cell counts based on foremilk sampling

Determination of somatic cell count (SCC) is used worldwide in dairy practice to describe the hygienic status of the milk and the udder health of cows. When SCC is tested on a quarter level to detect single quarters with high SCC levels of cows for practical reasons, mostly foremilk samples after prestimulation (i.e. cleaning of the udder) are used. However, SCC is usually different in different milk fractions. Therefore, the goal of this study was the investigation of the use of foremilk samples for the estimation of total quarter SCC. A total of 378 milkings in 19 dairy cows were performed with a special milking device to drain quarter milk separately. Foremilk samples were taken after udder stimulation and before cluster attachment. SCC was measured in foremilk samples and in total quarter milk. Total quarter milk SCC could not be predicted precisely from foremilk SCC measurements. At relatively high foremilk SCC levels (>300 x 10(3) cells/ml) foremilk SCC were higher than total quarter milk. At around (50-300) x 10(3) cells/ml foremilk and total quarter SCC did not differ considerably. Most interestingly, if foremilk SCC was lower than 50 x 10(3) cells/ml the total quarter SCC was higher than foremilk SCC. In addition, individual cows showed dramatic variations in foremilk SCC that were not very well related to total quarter milk SCC. In conclusion, foremilk samples are useful to detect high quarter milk SCC to recognize possibly infected quarters, only if precise cell counts are not required. However, foremilk samples can be deceptive if very low cell numbers are to be detected.

K1 protein of human herpesvirus 8 suppresses lymphoma cell Fas-mediated apoptosis.

Expression of the K1 gene of human herpesvirus 8 activates nuclear factor-kappaB and induces lymph node hyperplasia and lymphomas in transgenic mice. To further delineate its role in cell survival, we determined whether K1 altered apoptosis of lymphoma cells. K1 protein is expressed in Kaposi sarcoma and primary effusion lymphoma. We retrovirally transfected BJAB lymphoma, THP-1, U937, and Kaposi sarcoma SLK cells to express K1 and a K1 mutant with the deleted immunoreceptor tyrosine-based activation motif (K1m). We challenged cells with an agonistic anti-Fas antibody, Fas ligand, irradiation, and tumor necrosis factor-related apoptosis-inducing ligand. K1 transfectants but not K1m transfectants exhibited reduced levels of apoptosis induced by the anti-Fas antibody but not apoptosis induced by the tumor necrosis factor-related apoptosis-inducing ligand or irradiation. K1 expression resulted in reduced apoptosis rates as shown in several assays. K1 induced a modest reduction in levels of Fas-associated death domain protein, and procaspase 8 recruited to the death-inducing signaling complex. Finally, K1 transfectants cleaved procaspase 8 at significantly lower rates than did K1m transfectants. K1-transfected mice, compared with vector-transfected mice, showed lower death rates after challenge with anti-Fas antibody. K1 may contribute to lymphoma development by stimulating cell survival by selectively blocking Fas-mediated apoptosis.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used. For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions. For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream kinases sensitive to changes in ATP levels, were also performed. The hypophosphorylated state of 4E-BP1 leads to translation repression. Data indicated that treatment with 17-AAG alone resulted in a minor (<10%) change in STAT3, Raf-1, and Akt protein levels, while no change was observed for 4E-BP1. The combination treatment resulted in more than 50% decrease of the client protein levels and hypophosphorylation of 4E-BP1 in all MM cell lines. Treatment with 8-Cl-Ado alone resulted in less than 30% decrease in client protein levels as well as a decrease in 4E-BP1 phosphorylation. Cytotoxic evaluation of 8-Cl-Ado in combination with 17-AAG resulted in more than additive cytotoxicity when drugs were combined in a sequential manner. In summary, these data suggest that the mechanism-based combination of agents that target transcription, translation, or decrease cellular bioenergy with 17-AAG results in increase cytotoxicity when compared to the single agents. Such combination strategies may be applied in the clinic since these drugs are established chemotherapeutic agents or currently in clinical trials.

PCR-based assay using occult blood detection cards for detection of diarrheagenic Escherichia coli in specimens from U.S. travelers to Mexico with acute diarrhea.

Large field studies of travelers' diarrhea for multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport, and storage of fecal specimens that does not require immediate processing and refrigeration and that is stable for months would be advantageous. This study was designed to determine if enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) DNA could be identified from cards that were processed for the evaluation of fecal occult blood. U.S. students traveling to Mexico during 2005 to 2007 were monitored for the occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard methods. Cards then were stored at room temperature prior to DNA extraction. Fecal PCR was performed to identify ETEC and EAEC in DNA extracted from stools and from occult blood cards. Significantly more EAEC cases were identified by PCR that was performed on DNA that was extracted from cards (49%) or from frozen feces (40%) than from culture methods that used HEp-2 adherence assays (13%) (P < 0.001). Similarly, more ETEC cases were detected from card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). The sensitivity and specificity of the card test were 75 and 62%, respectively, compared to those for EAEC by culture and were 50 and 63%, respectively, compared to those for ETEC. DNA extracted from fecal cards that was used for the detection of occult blood is of use in identifying diarrheagenic E. coli.

The Role of Cell Sterilization in Population Based Studies of Radiogenic Second Cancers Following Radiation Therapy

Advances in radiotherapy have generated increased interest in comparative studies of treatment techniques and their effectiveness. In this respect, pediatric patients are of specific interest because of their sensitivity to radiation induced second cancers. However, due to the rarity of childhood cancers and the long latency of second cancers, large sample sizes are unavailable for the epidemiological study of contemporary radiotherapy treatments. Additionally, when specific treatments are considered, such as proton therapy, sample sizes are further reduced due to the rareness of such treatments. We propose a method to improve statistical power in micro clinical trials. Specifically, we use a more biologically relevant quantity, cancer equivalent dose (DCE), to estimate risk instead of mean absorbed dose (DMA). Our objective was to demonstrate that when DCE is used fewer subjects are needed for clinical trials. Thus, we compared the impact of DCE vs. DMA on sample size in a virtual clinical trial that estimated risk for second cancer (SC) in the thyroid following craniospinal irradiation (CSI) of pediatric patients using protons vs. photons. Dose reconstruction, risk models, and statistical analysis were used to evaluate SC risk from therapeutic and stray radiation from CSI for 18 patients. Absorbed dose was calculated in two ways: with (1) traditional DMA and (2) with DCE. DCE and DMA values were used to estimate relative risk of SC incidence (RRCE and RRMA, respectively) after proton vs. photon CSI. Ratios of RR for proton vs. photon CSI (RRRCE and RRRMA) were then used in comparative estimations of sample size to determine the minimal number of patients needed to maintain 80% statistical power when using DCE vs. DMA. For all patients, we found that protons substantially reduced the risk of developing a second thyroid cancer when compared to photon therapy. Mean RRR values were 0.052±0.014 and 0.087±0.021 for RRRMA and RRRCE, respectively. However, we did not find that use of DCE reduced the number of patents needed for acceptable statistical power (i.e, 80%). In fact, when considerations were made for RRR values that met equipoise requirements and the need for descriptive statistics, the minimum number of patients needed for a micro-clinical trial increased from 17 using DMA to 37 using DCE. Subsequent analyses revealed that for our sample, the most influential factor in determining variations in sample size was the experimental standard deviation of estimates for RRR across the patient sample. Additionally, because the relative uncertainty in dose from proton CSI was so much larger (on the order of 2000 times larger) than the other uncertainty terms, it dominated the uncertainty in RRR. Thus, we found that use of corrections for cell sterilization, in the form of DCE, may be an important and underappreciated consideration in the design of clinical trials and radio-epidemiological studies. In addition, the accurate application of cell sterilization to thyroid dose was sensitive to variations in absorbed dose, especially for proton CSI, which may stem from errors in patient positioning, range calculation, and other aspects of treatment planning and delivery.

Identification of cadmium-induced mutations in a mammalian cell line

Epidemiological studies have shown cadmium to induce cancer in humans, while experimental studies have proven this metal to be a potent tumor inducer in animals. However, cadmium appears nonmutagenic in most prokaryotic and eukaryotic mutagenesis assays. In this study, we present the identification of mutations in normal rat kidney cells infected with the mutant MuSVts110 retrovirus (6m2 cells) as a result of treatment with cadmium chloride. The detection of these mutations was facilitated by the use of a novel mutagenesis assay established in this laboratory. The 6m2 reversion assay is a positive selection system based on the conditional expression of the MuSVts110 v-mos gene. In MuSVts110 the gag and mos genes are fused out of frame, thus the translation of the v-mos sequence requires a frameshift in the genomic RNA. In 6m2 cells this frameshift is accomplished by the temperature-dependent splicing of the primary MuSVts110 transcript. Splicing of MuSVts110, which is mediated by cis-acting sequences, occurs when 6m2 cells are grown at 33$\sp\circ$C and below, but not at 39$\sp\circ$C. Therefore, 6m2 cells appear transformed at low growth temperatures, but take on a morphologically normal appearance when grown at high temperatures. The treatment of 6m2 cells with cadmium chloride resulted in the outgrowth of a number of cells that reverted to the transformed state at high growth temperatures. Analysis of the viral proteins expressed in these cadmium-induced 6m2 revertants suggested that they contained mutations in their MuSVts110 DNA. Sequencing of the viral DNA from three revertants that constitutively expressed the P85$\sp{gag{-}mos}$ transforming protein revealed five different mutations. The Cd-B2 revertant contained three of those mutations: an A-to-G transition 48 bases downstream of the MuSVts110 3$\sp\prime$ splice site, plus a G-to-T and an A-to-T transversion 84 and 100 bases downstream of the 5$\sp\prime$ splice site, respectively. The Cd-15-5 revertant also contained a point mutation, a T-to-C transition 46 bases downstream of the 5$\sp\prime$ splice site, while Cd-10-5 contained a three base deletion of MuSVts110 11 bases upstream of the 3$\sp\prime$ splice site. A fourth revertant, Cd-10, expressed a P100$\sp{gag{-}mos}$ transforming protein, and was found to have a two base deletion. This deletion accomplished the frameshift necessary for v-mos expression, but did not alter MuSVts110 RNA splicing and the expression of p85$\sp{gag{-}mos}.$ Lastly, sequencing of the MuSVts110 DNA from three spontaneous revertants revealed the same G to T transversion in each one. This was the same mutation that was found in the Cd-B2 revertant. These findings provide the first example of mutations resulting from exposure to cadmium and suggest, by the difference in each mutation, the complexity of the mechanism utilized by cadmium to induce DNA damage. ^

Cell force measurements in 3D microfabricated environments based on compliant cantilevers.

We report the fabrication, functionalization and testing of microdevices for cell culture and cell traction force measurements in three-dimensions (3D). The devices are composed of bent cantilevers patterned with cell-adhesive spots not lying on the same plane, and thus suspending cells in 3D. The cantilevers are soft enough to undergo micrometric deflections when cells pull on them, allowing cell forces to be measured by means of optical microscopy. Since individual cantilevers are mechanically independent of each other, cell traction forces are determined directly from cantilever deflections. This proves the potential of these new devices as a tool for the quantification of cell mechanics in a system with well-defined 3D geometry and mechanical properties.

Towards personalized medicine: Chemosensitivity assays of patient lung cancer cell spheroids in a perfused microfluidic platform

Cancer is responsible for millions of deaths worldwide and the variability in disease patterns calls for patient-specific treatment. Therefore, personalized treatment is expected to become a daily routine in prospective clinical tests. In addition to genetic mutation analysis, predictive chemosensitive assays using patient's cells will be carried out as a decision making tool. However, prior to their widespread application in clinics, several challenges linked to the establishment of such assays need to be addressed. To best predict the drug response in a patient, the cellular environment needs to resemble that of the tumor. Furthermore, the formation of homogeneous replicates from a scarce amount of patient's cells is essential to compare the responses under various conditions (compound and concentration). Here, we present a microfluidic device for homogeneous spheroid formation in eight replicates in a perfused microenvironment. Spheroid replicates from either a cell line or primary cells from adenocarcinoma patients were successfully created. To further mimic the tumor microenvironment, spheroid co-culture of primary lung cancer epithelial cells and primary pericytes were tested. A higher chemoresistance in primary co-culture spheroids compared to primary monoculture spheroids was found when both were constantly perfused with cisplatin. This result is thought to be due to the barrier created by the pericytes around the tumor spheroids. Thus, this device can be used for additional chemosensitivity assays (e.g. sequential treatment) of patient material to further approach the personalized oncology field.