RESUMO
Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.
Assuntos
Glucose , Animais , Cricetinae , Proliferação de Células , Células CHO , Cricetulus , Proteínas Recombinantes/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Low temperature and sodium butyrate (NaBu) are two of the most used productivity-enhancing strategies in CHO cell cultures during biopharmaceutical manufacturing. While these two approaches alter the balance in the reciprocal relationship between cell growth and productivity, we do not fully understand their mechanisms of action beyond a gross cell growth inhibition. Here, we used continuous culture to evaluate the differential effect of low temperature and NaBu supplementation on CHO cell performance and gene expression profile. We found that an increase in cell-productivity under growth-inhibiting conditions was associated with the arrest of cells in the G1/G0 phase. A transcriptome analysis revealed that the molecular mechanisms by which low temperature and NaBu arrested cell cycle in G1/G0 differed from each other through the deregulation of different cell cycle checkpoints and regulators. The individual transcriptome changes in pattern observed in response to low temperature and NaBu were retained when these two strategies were combined, leading to an additive effect in arresting the cell cycle in G1/G0 phase. The findings presented here offer novel molecular insights about the cell cycle regulation during the CHO cell bioprocessing and its implications for increased recombinant protein production. This data provides a background for engineering productivity-enhanced CHO cell lines for continuous manufacturing.
Assuntos
Técnicas de Cultura de Células , Cricetinae , Animais , Células CHO , Fase de Repouso do Ciclo Celular , Cricetulus , Proteínas Recombinantes/metabolismo , Ciclo CelularRESUMO
In the biopharmaceutical sector, Chinese hamster ovary (CHO) cells have become the host of choice to produce recombinant proteins (r-proteins) due to their capacity for correct protein folding, assembly, and posttranslational modification. However, the production of therapeutic r-proteins in CHO cells is expensive and presents insufficient production yields for certain proteins. Effective culture strategies to increase productivity (qp) include a high glucose concentration in the medium and mild hypothermia (28-34 °C), but these changes lead to a reduced specific growth rate. To study the individual and combined impacts of glucose concentration, specific growth rate and mild hypothermia on culture performance and cell metabolism, we analyzed chemostat cultures of recombinant human tissue plasminogen activator (rh-tPA)-producing CHO cell lines fed with three glucose concentrations in feeding media (20, 30 and 40 mM), at two dilution rates (0.01 and 0.018 1/h) and two temperatures (33 and 37 °C). The results indicated significant changes in cell growth, cell cycle distribution, metabolism, and rh-tPA productivity in response to the varying environmental culture conditions. High glucose feed led to constrained cell growth, increased specific rh-tPA productivity and a higher number of cells in the G2/M phase. Low specific growth rate and temperature (33 °C) reduced glucose consumption and lactate production rates. Our findings indicated that a reduced specific growth rate coupled with high feed glucose significantly improves r-protein productivity in CHO cells. We also observed that low temperature significantly reduced qp, but not cell growth when dilution rate was manipulated, regardless of the glucose concentration or dilution rate. In contrast, we determined that feed glucose concentration and consumption rate were the dominant aspects of the growth and productivity in CHO cells by using multivariate analysis.
Assuntos
Proliferação de Células/efeitos dos fármacos , Temperatura Baixa , Glucose/farmacologia , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Cricetulus , Humanos , Hipotermia , Análise de Componente Principal , Proteínas Recombinantes/genética , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/genéticaRESUMO
Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.
Assuntos
Anticorpos Monoclonais/metabolismo , Biotecnologia/métodos , Técnicas de Cultura de Células/métodos , Sobrevivência Celular/fisiologia , Temperatura Baixa , Animais , Células CHO , Proliferação de Células/fisiologia , Cricetinae , Cricetulus , Humanos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
INTRODUCTION: Brain tumors are present in 2.9 per 100,000 newborn. Craniopharyngioma is a benign and slow growing brain tumor, frequently localized in the sellar and suprasellar region. There are few reports of pituitary tumor detected prenatally. CASE REPORT: We report a neonate with a craniopharyngioma detected prenatally as a pituitary tumor. In a 23 year old mother, second gestation, with no important history, was detected a sellar tumor at 31 gestation weeks, the obstetric ultrasound reported a suprasellar tumor of 2 per 3 cm diameter. Pregnancy ended in a vaginal delivery at 39 weeks, and obtained a 3.9 kg female, with cephalic diameter of 37.5 cm, the Apgar score was 8-9 at 1st and 5th minutes. In early neonatal period was scanned and confirmed a 3.2/2.3/2.9 cm suprasellar tumor with calcium deposits. The Paediatric Oncology department suggested a surgery and was realized a craniotomy at 3rd week of age. The surgery allowed to obtain 30% of the tumor and confirmed by histology craniopharyngioma. Patient had favourable evolution and was discharged at 3 months of age. CONCLUSIONS: We report a neonate in who was detected by prenatal ultrasound the presence of a suprasellar solid tumor, scan and magnetic resonance images in neonatal period defined its size and location and a craniopharyngioma was confirmed by histology. Patient had a satisfactory postsurgical evolution and was discharged at 3 months of age.
Assuntos
Craniofaringioma/congênito , Neoplasias Hipofisárias/congênito , Ultrassonografia Pré-Natal , Craniofaringioma/diagnóstico por imagem , Craniofaringioma/embriologia , Craniofaringioma/cirurgia , Craniotomia , Feminino , Humanos , Hipofisectomia/métodos , Recém-Nascido , Imageamento por Ressonância Magnética , Neoplasias Hipofisárias/diagnóstico por imagem , Neoplasias Hipofisárias/embriologia , Neoplasias Hipofisárias/cirurgia , Indução de Remissão , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
UNLABELLED: There are few reports of prenatal diagnosis of severe pulmonary valvar stenosis (PVS). It affects 1/22,000 newborn and represents 8-10% of total congenital cardiac defects. Clinic CASE: we report a case of a neonate in which was prenatally detected a pulmonary valvar stenosis and was successfully corrected with early valvuloplasty. From a 36-Year-old woman sent to evaluation to the fetal maternal unit because a tricuspid valvar insufficiency detected at 36 gestation weeks (GW). A VPS was suspected before born and a pregnancy ended in programated caesarean delivery at 38 GW, obtaining a 3 kg male, in which early echocardiography reported a severe PVS, promptly was initiated prostaglandin E1 (PgE1) infusion avoiding patent ductus arteriosus (PDA) closure, following a percutaneus balloon dilatation valvuloplasty at 48 hours, improving cyanosis and transvalvular Doppler flow. CONCLUSION: we report a neonate referred with an opportune prenatal diagnosis of tricuspid insufficiency and confirmed a severe PVS, PgE1 was infused immediately after born, allowing successfully balloon dilatation valvuloplasty in first 48 hours.
Assuntos
Cateterismo , Estenose da Valva Pulmonar/diagnóstico por imagem , Estenose da Valva Pulmonar/terapia , Ultrassonografia Pré-Natal , Adulto , Feminino , Humanos , Recém-NascidoRESUMO
Polycystic kidney disease is a common genetic cause of chronic kidney disease, characterized by the formation of multiple cysts in the kidneys and other organs, occurs in 1 in 20,000 live births. 30 to 50% of affected newborns die shortly after birth because of respiratory and renal insufficiency. This study reports the case of a newborn with polycystic kidney disease diagnosed by obstetric ultrasound at 26 weeks of gestation and kidneys anhidramnios due to increased volume and appearance "in sponge." Neonato a primigravida 19 years of age. At 26 weeks you will be detected in a routine obstetric evaluation that measures were in line for somatométricas fetal gestational age, and anhidramnios increase in renal mass and bilateral thorax narrow. The pregnancy ended in a cesarean section at 37 weeks with a newborn of 3140 g, women who died within minutes after birth. We requested an autopsy because of the need for genetic counseling. The findings were: enlarged kidneys with microcystic dilatation of collecting duct and in the renal cortex and medulla, liver fibrosis and Müllerian duplication with double uterine cavity. It is a rare association between polycystic kidney disease and Müllerian duplication, liver fibrosis confirmed by autopsy and has not been documented previously.
Assuntos
Ductos Paramesonéfricos/anormalidades , Ductos Paramesonéfricos/diagnóstico por imagem , Doenças Renais Policísticas/diagnóstico por imagem , Ultrassonografia Pré-Natal , Humanos , Recém-NascidoRESUMO
BACKGROUND: Multiple pregnancies prevalence has been increasing in last decade, which have also increased the requirements of neonatal intensive care units and all problems related to premature neonate or low birth weight. Prevalence rate of twin (18 to 26 in 1,000 births), and triple pregnancies (0.37 to 1.74 in 1,000 births) have raised too, perhaps due to assisted reproductive techniques. OBJECTIVE: To know incidence of multiple pregnancies at Unidad Medica de Alta Especialidad no. 23, from Institute Mexicano del Seguro Social. MATERIAL AND METHOD: Retrospective and descriptive study. We review the files of multiple pregnancies from 1972 to 2006 to estimate its rate and change every five and ten years. RESULTS: We registered 9,055 twin pregnancies during the period, with a rate of 7.1 to 14.4 in 1,000 (63% of increase in the last decade [12.6 in 1,000 births] compared with the previous decade [7.7 in 1,000 births]; p < 0.005). Pregnancies with three or more fetuses were 202, with 191 triplets, 13 with four, three with five, and one with six products (646 newborns). Incidence of multiple pregnancies with four or more products has also increased in last decade: 230 times higher than two decades before. CONCLUSION: Multiple pregnancies rate has increased in last decade: 63% in twin pregnancies, 217% in triplets, and 230 times more than expected in four or more products pregnancies.
Assuntos
Gravidez Múltipla/estatística & dados numéricos , Feminino , Humanos , Incidência , Gravidez , Prevalência , Estudos RetrospectivosRESUMO
BACKGROUND: pregnant women with Rh alloimmunization (RhA) are submitted to invasive procedures to assess fetal anemia (FA). Recently a non-invasive approach to FA diagnosis has been proposed using Doppler ultrasound (DU) to identify increased peak velocity of systolic blood flow (Vm) in the middle cerebral artery (MCA). METHODOLOGY: eleven Rh alloimmunized pregnant women with serum red-cell antibody titers > 1:16 were included. Twenty-four procedures were done measuring the VmMCA followed by cordocentesis and fetal hemoglobin (FH) analysis. Pearson's linear correlation was calculated between the multiples of the median (MoM) of the VmMCA and the MoM of the FH, as well as the sensitivity, specificity and positive predictive value (PPV) for FA prediction. RESULTS: we found FA (FH mean = 6 g/dL) in 12 of 24 evaluations with a VmMCA mean of 1.5 MoM and a range from 1.22 to 1.68 MoM; in the remaining 12 cases the FH was normal (FH mean = 13.1 g/dL) with a VmMCA mean of 0.97 MoM and a range from 0.35-1.17 MoM (p < 0.001). Eleven fetuses with anemia had a MoM of the VmMCA above 1.29, except one with 1.22 MoM. The linear correlation between the MoM of the VmMCA and the MoM of FH was 0.83. The sensitivity of the MoM of the VmMCA to detect FA was 91%, specificity of 100% and PPV of 100%. CONCLUSIONS: DU measurement of the VmMCA was a useful non-invasive technique to evaluate FA. The sensitivity and PPV for FA diagnosis in RhA was above 90%.
Assuntos
Anemia/sangue , Anemia/diagnóstico por imagem , Doenças Fetais/sangue , Doenças Fetais/diagnóstico por imagem , Isoimunização Rh , Ultrassonografia Doppler , Ultrassonografia Pré-Natal , Feminino , Humanos , Valor Preditivo dos Testes , Gravidez , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: The prevalence of congenital cardiac defects is 8 per 1000 neonates, and it's different if high or low risk populations are studied. The fetal ultrasonographic increase prenatal detection but varies from 7 to 90%. OBJECTIVES: To know the prevalence of fetal cardiopathy and detection in high risk pregnancies. PATIENTS AND METHODS: A observational study was made in pregnancies women with 16 old week of gestation. RESULTS: We received a total of 3500 high-risk pregnancies and were detected 112 cases with fetal cardiopathy (3.2%). The 30% of them had a risk factor of cardiopathy. The most frequent fetal cardiac defect detected were arrhythmia in 34 fetus, septal defects in 30, valvular defects in 17, hypoplasic or absence of cardiac cavities 16, tronco-conus defects 8, and other 7 included ectopia cordis 3, cardiac tumor 2, abnormal drainage of pulmonary veins 2. The diagnosis increased every year since started study. The prenatal diagnoses suspected in fetal echocardiography were confirmed in 80% of the cases in neonatal period. CONCLUSION: The detection rate of fetal cardiac defect was 3.2% in high-risk pregnancies, four times higher than general population prevalence of congenital heart disease. We found a 30% overall perinatal mortality in fetal cardiac defect. The most frequent fetal cardiac defects found in this screening were arrhythmias and septal ventricular defects in almost 50% of patients.