This study investigates the efficacy of a newly developed humanized serum-free media (SFM) that replaces Fetal Bovine Serum (FBS), in culturing of common in vitro models (Caco-2 and 3D Primary Human Hepatocytes (PHH)). The research’s focus aligns with the 3R principles (replacement, reduction, refinement) and may potentially lead to enhancing the human relevance of the in vitro models and ultimately contribute to more accurate and successful drug development in the future. This study is divided into two subprojects, Caco-2 and 3D PHH, respectively. For Caco-2, we evaluated cell morphology, proliferation and monolayer formation by assessing the permeability of marker compounds. For PHH, we investigated spheroid formation and viability using an ATP measurement assay, lipid concentration using an Adipored Assay and CYP activity of the 3D PHH. Finally, global proteomics analysis was performed for both Caco-2 cells and 3D PHH grown in SFM and in conventional medium (CM). Our findings show that Caco-2 cells could proliferate well enough in the SFMCaco-2 without any change in their cell morphology, whereas their monolayer tightness was found to be similar in SFMCaco-2 as compared to CMCaco-2. Furthermore, from the proteomics analysis there was no major difference detected between cells in SFMCaco-2 and in CMCaco-2, however several of the relevant drug transporters of this model were found in higher levels in Caco-2 cultured in SFMCaco-2. For 3D PHH, our findings indicate that spheroids could be perfectly formed in both SFM3D PHH and in CM3D PHH, having relatable viability and lipid concentration, especially after two week of culturing period. Finally, the proteomics analysis revealed no significant difference between the proteome of the spheroids when cultured in SFM3D PHH or in CM3D PHH. In conclusion, these results suggest that the SFM provides a viable and sometimes superior alternative to CM for both Caco-2 and 3D PHH, promoting the 3R principles and potentially offering a more human-relevant model for in vitro studies.

Orientadores: Simone Cristina Olenscki Gilli, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T07:27:16Z (GMT). No. of bitstreams: 1 Simoneti_GiseledaSilva_M.pdf: 1770601 bytes, checksum: 967e921fb162c153636ac91afdd1f138 (MD5) Previous issue date: 2013 / Resumo: Células dendríticas (DCs) são as principais células apresentadoras de antígeno do sistema imune, capazes de estimular o linfócito T a iniciar resposta imune especifica. Vacinas de DCs vêm sendo utilizadas como forma de tratamento imunoterápico adjuvante para várias neoplasias. Protocolos para geração dessas células têm sido desenvolvidos e o método ideal de produção para uso clínico ainda necessita ser definido. É fundamental a definição de protocolos e reagentes que ofereçam, a partir de células mononucleares do sangue periférico, células dendríticas seguras e funcionais para uso clínico. A suplementação de meios de cultura com soro de origem animal e humano leva á riscos de xenosensibilização e transmissão de doenças. O uso do soro autólogo parece oferecer menos riscos ao paciente, porém a presença de fatores imunossupressores nesse soro poderia interferir na qualidade das DCs produzidas. Vários tipos de meios livres de soro, baseados nas boas práticas de produção - "good manufacture practice" (GMP), têm sido utilizados recentemente e parecem ser uma opção viável. O objetivo desse estudo foi avaliar os resultados da diferenciação, maturação e funcionalidade de DCs de pacientes com LMA, produzidas em meios livres de soro e em meio suplementado com soro autólogo. Concluímos que os meios de cultura livres de soro foram eficientes na produção de DCs para fins imunoterápicos em pacientes com LMA. Em contrapartida, o uso de soro autólogo parece interferir na capacidade funcional das DCs geradas / Abstract: Dendritic cells (DCs) are the main antigen-presenting cells of the immune system, capable of stimulating T lymphocytes to initiate specific immune responses. Vaccines based on DCs have been used as a treatment adjuvant immunotherapy for various malignancies. Protocols for generating these cells have been developed and the optimal method of production for clinical use remains to be defined. There is a great interest in the definition of protocols and reagents providing from peripheral blood mononuclear cells, functional and safe dendritic cells for clinical use. Supplementation of culture media with serum from animal and human leads to reactions due the animal proteins and transmission of disease. The use of autologous serum seems to offer less risk to the patient, but the presence of immunosuppressive factors may affect the quality of the DCs produced. Several types of serum-free media, based on "good manufacture practice" (GMP), have been used recently and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation and function of DCs from AML patients, generated in serum-free media and media supplemented with autologous serum. We concluded that the serum-free media were efficient in the production of DCs for immunotherapy in AML patients. However, the use of autologous serum appears to interfere with the functional capacity of generated DCs / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestra em Fisiopatologia Médica

No description available.

Thesis (MMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: Serum free light chains (FLC) are associated with imbalances in heavy and light chain production. Abnormal FLC ratios have been associated with risk of progression in certain diseases. Automated assays are available for their determination and they are used in the followup and management of patients with monoclonal gammopathies. Acceptable imprecision, specificity, accuracy and reproducibility between reagent batches is required to prevent under- or overestimation. Method validation is a standard process in every good laboratory to judge the acceptability of a new method. Reference intervals have been established in an older population, but it was considered important to verify these in our population. HIV is associated with B-cell dysfunction. As B-cell abnormalities are associated with disorders leading to monoclonal gammopathies, we postulated that the FLC levels and FLC ratio would be abnormal in HIV infected individuals. Methods and materials: Controls and pooled patient samples were used for the method validation study which included imprecision studies, linearity, recovery and interference studies, and method comparison studies, the latter compared our method to the same method used in another laboratory. For the reference interval study, blood was obtained from 120 healthy subjects. The following blood tests were performed: total protein, IgG, IgA, IgM, creatinine, protein electrophoresis, kappa FLC and lambda FLC. Using the kappa and lambda FLC results, a FLC ratio was determined. Three hundred and sixty-nine HIV positive subjects were then studied. The same tests were performed, as well as CD4+ counts and viral loads on the majority of them. Results: For the method validation study, precision, linearity and recovery was acceptable. Minimal interference was observed with haemolysis, lipaemia, bilirubin and rheumatoid factor. Our method showed comparable performance with the established method. For the reference interval study, all the creatinine values were normal, as were serum protein values. The serum protein electrophoreses were independently reviewed by 3 pathologists. Most were normal, with a few polyclonal increases seen, but no definite monoclonal bands. The 95% reference intervals for FLC’s as well as the FLC ratio were not statistically significantly different to the manufacturer’s recommendations. When examining the HIV positive study population, we found that FLC and FLC ratio were influenced by markers of HIV disease severity, such as CD4+ count, IgG, viral load, use of antiretroviral treatment and abnormal serum protein electrophoreses. Conclusion: The validation study of FLC showed excellent precision, acceptable bias, good linearity, good recovery and minimal interference, allowing routine introduction of the test. The 95% reference intervals obtained for our population were slightly higher than those recommended by the manufacturer. However, as most of the values fell within the manufacturer’s limits, we could accept the manufacturer’s recommended cut-offs. We found that FLC levels were definitely influenced by markers of HIV disease severity in our population and we postulate that they may be of use for follow-up of patients with HIV. / AFRIKAANSE OPSOMMING: Agtergrond: Serum vry ligte kettings (VLK) word geassosieer met ‘n wanbalans van ligte en swaar ketting produksie. Abnormale VLK ratios is geassosieer met ‘n risiko van verloop in sekere siektes. Geoutomatiseerde laboratorium toetse vir VLK is beskikbaar vir hul bepaling en word gebruik om pasiënte met monoklonale gammopatieë op te volg en te behandel. Aanvaarbare impresisie, spesifisiteit, akkuraatheid en herhaalbaarheid tussen reagens besendings is belangrik om onder- of oorbepaling te verhoed. Metode validasie is ’n standaard proses in elke goeie laboratorium om die aanvaarbaarheid van ’n nuwe metode te bepaal. Verwysingswaardes is al bepaal in ’n ouer populasie. Ons het besluit om die verwysingswaardes in ons populasie te bepaal. Mens-immuungebrekvirus (MIV) word geassosieer met B-sel disfunksie. Omdat B-sel abnormaliteite geassosieer word met afwykings wat tot monoklonale gammopatieë lei, het ons gepostuleer dat die VLK vlakke en VLK ratio abnormaal sal wees in MIV geïnfekteerde persone. Metodes en Materiale: Kontroles en pasiënt monsters is gebruik vir die metode validasie studie wat impresisie studies, lineariteit, herwinning, inmenging en metode korrelasie studies ingesluit het. In laasgenoemde geval is ons metode met dieselfde metode van ’n ander laboratorium vergelyk. Vir die verwysingswaardes studie is 120 gesonde persone se bloed gebruik. Die volgende toetse is bepaal: totale proteïen, IgG, IgA, IgM, kreatinien, proteïen elektroferese, kappa en lambda VLK. Die VLK ratio is bepaal deur die kappa en lambda resultate te gebruik. Driehonderd nege en sestig MIV-positiewe pasiente is gebruik vir die studie. Dieselfde toetse was gedoen, asook CD4+ tellings en virale ladings op die meerderheid van pasiente. Resultate: Vir die metode validasie studie, was presisie, lineariteit en herwinning aanvaarbaar. Minimale inmenging van hemolise, lipemie, bilirubien en rumatoïede factor is waargeneem. Ons metode het goed gekorreleer met die bepaalde metode. Die serum kreatinien en serum totale proteïen waardes was normaal tydens die verwysingswaardes studie. Die serum proteïen elektroferese was onafhanklik beoordeel deur 3 patoloë. Die meeste was normaal met enkele poliklonale verhogings, maar geen definitiewe monoklonale bande nie. Die 95% verwysings intervalle vir VLK en VLK ratio het nie statisties betekenisvol verskil van die vervaardiger se aanbevelings nie. In die studie van die MIV-positiewe studie populasie, het ons gevind dat VLK en VLK ratio beïnvloed word deur merkers van ernstige MIV siekte, soos CD4+ telling, IgG, virale lading, die gebruik van antiretrovale medikasie en abnormale serum proteïen elektroferese. Gevolgtrekking: Die validasie studie van VLK het uitstekende presisie, aanvaarbare partydigheid, goeie lineariteit, goeie herwinning en minimale inmenging gewys, wat die roetine instelling van die toets toegelaat het. Die 95% verwysingsintervalle wat vir ons populasie bepaal is, was effens hoër as die vervaardiger se aanbeveling. Die meeste van die waardes het egter binne die vervaardiger se limiete geval, dus kon ons die vervaardiger se afsnypunte aanvaar. Ons het gevind dat VLK vlakke definitief beïnvloed word deur merkers van die ernstigheidsgraad van MIV siekte in ons populasie en ons postuleer dat VLK van waarde kan wees met die opvolg van MIV pasiente. / NHLS / Harry Crossley for funding obtained

<p>The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells.</p><p>Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation.</p><p>Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics.</p><p>In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity.</p><p>Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well.</p>

La thérapie cellulaire est une méthode d’avenir innovante, actuellement utilisée dans le traitement de pathologies multiples (auto-immunitaire, cancéreuses, pathologies inflammatoires, allogreffes…) et la régénération des pertes de substance tissulaire. Les cellules souches mésenchymateuses, par la variabilité de leurs origines, présentent des propriétés très intéressantes à la thérapie, notamment un potentiel de différenciation en lignées multiples, et des propriétés d’immunomodulation importantes. Mon projet s’intéresse à l’utilisation de cellules souches orales récemment isolées de la gencive par notre équipe : cellules souches gingivales (GSC), et présentant un avantage fonctionnel par rapport aux sources cellulaires traditionnelles d’origine mésodermique (moelle osseuse) ou orales (pulpe dentaire, follicule dentaire, ligament parodontal, glandes salivaires…). Les défauts osseux des mâchoires, de par leur multitude d’étiologies (traumatismes, dysmorphoses, cancer, ...) et le handicap généré, représentent une cible thérapeutique privilégiée. Les GSCs ont la même origine embryologique neurectodermique que les os maxillaires et par là-même un phénotype proche, exploré dans notre équipe. Cette source gingivale de prélèvement non traumatique est une alternative aux techniques chirurgicales actuelles mutilantes pour le site donneur. Notre objectif est double : Etablir un protocole préclinique de culture des GSC en ostéoblastes, pour être compatibles avec la thérapie humaine afin d’obtenir une régénération osseuse optimale. Les capacités immunomodulatrices des GSCs sont par là-même étudiées dans ces nouvelles conditions, dans le but de maitriser la réaction inflammatoire et préserver la greffe osseuse, grâce à la plateforme exceptionnelle mise à notre disposition par l’établissement français du sang, et une équipe très spécialisée dans l’étude des mécanismes de régulation immunitaires. Nos résultats permettront non seulement une régénération osseuse transposable chez l’homme, mais également d’utiliser ces cellules pour le traitement d’autres pathologies (cancéreuses, auto-immunitaires…) en utilisant leur capacité immunomodulatrice. / Cell therapy is an innovative method of the future, currently used in the treatment of multiple diseases (autoimmune, cancer, inflammatory pathologies, allografts ...) and the regeneration of tissue loss. Mesenchymal stem cells (MSC), regardless their origins, exhibit very interesting properties for therapy, including a potential for multi-line differentiation, and important immunomodulation properties. My project focuses on the use of oral stem cells recently isolated from the gingiva by our team (GSC), and having a functional advantage over traditional mesodermal (bone marrow) cellular sources. The bone defects of the jaws, due to their multitude of etiologies (trauma, dysmorphoses, cancer...) and the generated handicap, represent a preferred therapeutic target. GSCs have the same neurectodermal embryological origin as the maxillary bones and thus a similar phenotype, explored in our team. This gingival source of non-traumatic removal is an alternative to current mutilating surgical techniques for the donor site. Our goal is twofold: To establish a preclinical GSC culture protocol in osteoblasts, to be compatible with human therapy, in order to achieve optimal bone regeneration. The immunomodulatory capacities of the GSCs are themselves studied under these new conditions, with the aim of controlling the inflammatory reaction and preserving the bone graft, thanks to the exceptional platform made available to us by the French blood establishment, and A highly specialized team in the study of immune regulation mechanisms. Our results will not only allow transposable bone regeneration in humans but also use these cells for the treatment of other pathologies (cancerous, autoimmune ...) using their immunomodulatory capacity.

Células HEK293T e Huh7 foram adaptadas em meios livres de soro fetal bovino (SFM). Parâmetros metabólicos e de crescimento foram avaliados, além da expressão gênica heteróloga, utilizando um sistema de expressão que produz pseudo-partículas (ppHCV), derivadas do vírus da Leucemia Murina (MLV) e da Hepatite C (HCV). A adaptação foi realizada através de diluição sequencial para SFM. A linhagem HEK293T foi adaptada em dois SFM: Hybridoma-SFM e CHO-S-SFMII, a linhagem Huh7 foi adaptada nos quatro SFM escolhidos. O consumo de substratos para cada linhagem foi diferente entre os SFM, apesar de o crescimento celular ter sido semelhante. Para a análise da expressão gênica, três vetores foram co-transfectados em células HEK293T. Foi observado que para a produção de ppHCV, o tempo de coleta foi de 48 horas. O método de co-transfecção por lipofectamina produziu mais cópias de vírus, sendo que quantificações de 5,30x103 cópias RNA/&#956;L foram encontradas para vírus produzidos em células adaptadas no meio Hybridoma-SFM através de qRT-PCR. Estas ppHCV foram usadas para infectar células Huh7, células infectadas produziram cerca de 10 ng de proteína recombinante/106 células. / HEK 293-T and Huh7 cells were adapted in serum free mediu (SFM). Metabolic and growth parameters were assessed, as well as heterologous gene expression, using an expression system that produces pseudo-particles (ppHCV), derived from the murine leukemia virus (MLV), and Hepatitis C (HCV). The adaptation was performed by sequential dilution in SFM. The HEK- 293T line was adapted in two SFM: Hybridoma-SFM and CHO-S-SFMII, the Huh7 line was adapted in four chosen SFM. The consumption of substrates were different for each line in SFM, while cell growth was similar. For the analysis of gene expression, three vectors were co-transfected into HEK-293T cells. It was observed that for the production of ppHCV, the collection time was 48 hours. The method of co-transfection with lipofectamine produced more copies of the virus into the cells, 5,30 x103 RNA copies/&#956;L were found to virus produced in the cells adapted in Hybridoma- SFM, by qRT-PCR. These ppHCV were used to infect Huh 7, infected cells produced around 10 ng recombinant protein /106 cells.

A produção de proteínas recombinantes, principalmente para uso farmacêutico, tem sido intensamente estudada, juntamente com suas propriedades físico-químicas, o que possibilita uma melhor escolha da técnica de purificação, e assim, evitar perdas no rendimento e custos elevados. A glicocerebrosidase (GCR) é uma enzima lisossomal, e sua deficiência ocasiona um distúrbio autossômico recessivo denominado doença de Gaucher. Atualmente o tratamento para essa patologia é por meio da Terapia de Reposição Enzimática (TRE), a qual tem sido realizada com grande êxito. Tendo em vista fornecer dados que possam auxiliar na redução do número de etapas cromatográficas no processo de downstream, este trabalho teve como objetivo, a purificação da GCR expressa por células de Ovário de Hamster Chinês (CHO), em meio livre de soro fetal bovino (SFB). Para se alcançar tais resultados, realizou-se o cultivo das células CHO-GCR em meio quimicamente definido livre de SFB (CHO-S-SFM II). Foram realizadas três técnicas cromatográficas (troca iônica, interação hidrofóbica e afinidade), com base em suas propriedades físico-químicas. E para o ensaio da atividade enzimática, foi utilizado o substrato fluorogênico 4-Methylumbeliferyl &#946;-D-glucopyranoside (4MU-G). As células CHO-GCR cultivadas em meio CHO-S-SFM II, apresentando viabilidade maior que 95%, e produção da GCR ativa, durante todo o período de cultivo. Os protocolos aplicados para a purificação da GCR, não apresentaram resultados significativos. Com o volume não retido após cromatografia por interação hidrofóbica, se estimou os valores de KM 2,13 e VMAX 0,0295 UFR/h para as constantes cinéticas da GCR. A diálise no processo de purificação mostrou ser uma etapa necessária para a atividade enzimática da GCR. No cultivo das células CHO-GCR para a formação do banco de trabalho, nos meios RPMI 1640 e &#945;-MEM, ambos com a adição de 10% SFB, não houve diferença significativa no crescimento entre eles, e apresentaram 100% de viabilidade durante todo o período de cultivo. Porém, ao analisar de forma isolada a fase exponencial de cada curva, notou-se que às células cultivadas no meio RPMI 1640, apresentaram taxa de crescimento superior, às cultivadas em meio &#945;-MEM. Concluiu-se que a expressão da GCR em meio livre de SFB, proporciona amostras menos complexas, em relação aos meios de cultura que necessitam de suplementação com SFB, o que pode reduzir o número de etapas cromatográficas, melhorando o rendimento e a redução da perda da atividade da GCR. O meio basal RPMI 1640 com a adição de SFB foi uma alternativa satisfatória para o cultivo das células CHO-GCR. Este estudo forneceu dados que podem contribuir para a melhoria do processo de purificação da GCR. Novas pesquisas podem ser desenvolvidas a fim de melhorar o processo de purificação da GCR. / The production of recombinant proteins, mainly for pharmaceutical use, has been intensively studied along with its physico-chemical properties, which allows a better choice of the purification technique, and thus avoid losses in yield and high costs. Glucocerebrosidase (GCR) is a lysosomal enzyme, and its deficiency causes an autosomal recessive disorder called Gaucher\'s disease. Currently the treatment for this pathology is through Enzymatic Replacement Therapy (ERT), which has been successfully performed. In order to provide data that may help reducing the number of chromatographic steps in the downstream process, this work aimed to purify GCR expressed by Chinese Hamster Ovary (CHO) cells in fetal bovine serum free (FBS). To achieve such results, the CHO-GCR cells were cultured in chemically defined Serum-free medium (CHO-S-SFM II). Three chromatographic techniques (ion exchange, hydrophobic interaction and affinity) were performed, based on their physicochemical properties. And for the enzymatic activity assay, the fluorogenic substrate 4-Methylumbeliferyl &#946;-D-glucopyranoside (4MU-G) was used. CHO-GCR cells cultured in CHO-S-SFM II medium, presenting viability greater than 95% and GCR production active, throughout the culture period. The protocols applied for GCR purification did not present significant results. With the volume not retained after chromatography by hydrophobic interaction, KM values of 2.13 and VMAX 0.0295 UFR/h were estimated for GCR kinetic constants. Dialysis in the purification process was shown to be a step necessary for the enzymatic activity of GCR. In the culture of the CHO-GCR cells for the formation of the working bank, in the media RPMI 1640 and &#945;-MEM, both with the addition of 10% FBS, there was no significant growth difference between them, and they showed 100% viability during all the growing period. However, when analyzing in isolation, the exponential phase of each curve, it was observed that the cells grown in RPMI 1640 medium showed higher growth rates, tham grown in &#945;-MEM medium. It was concluded that the expression of GCR in serum-free medium provides less complex samples, relative to the culture media requiring FBS supplementation, which may reduce the number of chromatographic steps, improving yield and loss reduction of GCR activity. The basal medium RPMI 1640 with the addition of FBS was a satisfactory alternative for culturing the CHO-GCR cells. This study provided data that may contribute to the improvement of the GCR purification process. New research can be developed to improve the GCR purification process.

A produção de proteínas recombinantes, principalmente para uso farmacêutico, tem sido intensamente estudada, juntamente com suas propriedades físico-químicas, o que possibilita uma melhor escolha da técnica de purificação, e assim, evitar perdas no rendimento e custos elevados. A glicocerebrosidase (GCR) é uma enzima lisossomal, e sua deficiência ocasiona um distúrbio autossômico recessivo denominado doença de Gaucher. Atualmente o tratamento para essa patologia é por meio da Terapia de Reposição Enzimática (TRE), a qual tem sido realizada com grande êxito. Tendo em vista fornecer dados que possam auxiliar na redução do número de etapas cromatográficas no processo de downstream, este trabalho teve como objetivo, a purificação da GCR expressa por células de Ovário de Hamster Chinês (CHO), em meio livre de soro fetal bovino (SFB). Para se alcançar tais resultados, realizou-se o cultivo das células CHO-GCR em meio quimicamente definido livre de SFB (CHO-S-SFM II). Foram realizadas três técnicas cromatográficas (troca iônica, interação hidrofóbica e afinidade), com base em suas propriedades físico-químicas. E para o ensaio da atividade enzimática, foi utilizado o substrato fluorogênico 4-Methylumbeliferyl &#946;-D-glucopyranoside (4MU-G). As células CHO-GCR cultivadas em meio CHO-S-SFM II, apresentando viabilidade maior que 95%, e produção da GCR ativa, durante todo o período de cultivo. Os protocolos aplicados para a purificação da GCR, não apresentaram resultados significativos. Com o volume não retido após cromatografia por interação hidrofóbica, se estimou os valores de KM 2,13 e VMAX 0,0295 UFR/h para as constantes cinéticas da GCR. A diálise no processo de purificação mostrou ser uma etapa necessária para a atividade enzimática da GCR. No cultivo das células CHO-GCR para a formação do banco de trabalho, nos meios RPMI 1640 e &#945;-MEM, ambos com a adição de 10% SFB, não houve diferença significativa no crescimento entre eles, e apresentaram 100% de viabilidade durante todo o período de cultivo. Porém, ao analisar de forma isolada a fase exponencial de cada curva, notou-se que às células cultivadas no meio RPMI 1640, apresentaram taxa de crescimento superior, às cultivadas em meio &#945;-MEM. Concluiu-se que a expressão da GCR em meio livre de SFB, proporciona amostras menos complexas, em relação aos meios de cultura que necessitam de suplementação com SFB, o que pode reduzir o número de etapas cromatográficas, melhorando o rendimento e a redução da perda da atividade da GCR. O meio basal RPMI 1640 com a adição de SFB foi uma alternativa satisfatória para o cultivo das células CHO-GCR. Este estudo forneceu dados que podem contribuir para a melhoria do processo de purificação da GCR. Novas pesquisas podem ser desenvolvidas a fim de melhorar o processo de purificação da GCR. / The production of recombinant proteins, mainly for pharmaceutical use, has been intensively studied along with its physico-chemical properties, which allows a better choice of the purification technique, and thus avoid losses in yield and high costs. Glucocerebrosidase (GCR) is a lysosomal enzyme, and its deficiency causes an autosomal recessive disorder called Gaucher\'s disease. Currently the treatment for this pathology is through Enzymatic Replacement Therapy (ERT), which has been successfully performed. In order to provide data that may help reducing the number of chromatographic steps in the downstream process, this work aimed to purify GCR expressed by Chinese Hamster Ovary (CHO) cells in fetal bovine serum free (FBS). To achieve such results, the CHO-GCR cells were cultured in chemically defined Serum-free medium (CHO-S-SFM II). Three chromatographic techniques (ion exchange, hydrophobic interaction and affinity) were performed, based on their physicochemical properties. And for the enzymatic activity assay, the fluorogenic substrate 4-Methylumbeliferyl &#946;-D-glucopyranoside (4MU-G) was used. CHO-GCR cells cultured in CHO-S-SFM II medium, presenting viability greater than 95% and GCR production active, throughout the culture period. The protocols applied for GCR purification did not present significant results. With the volume not retained after chromatography by hydrophobic interaction, KM values of 2.13 and VMAX 0.0295 UFR/h were estimated for GCR kinetic constants. Dialysis in the purification process was shown to be a step necessary for the enzymatic activity of GCR. In the culture of the CHO-GCR cells for the formation of the working bank, in the media RPMI 1640 and &#945;-MEM, both with the addition of 10% FBS, there was no significant growth difference between them, and they showed 100% viability during all the growing period. However, when analyzing in isolation, the exponential phase of each curve, it was observed that the cells grown in RPMI 1640 medium showed higher growth rates, tham grown in &#945;-MEM medium. It was concluded that the expression of GCR in serum-free medium provides less complex samples, relative to the culture media requiring FBS supplementation, which may reduce the number of chromatographic steps, improving yield and loss reduction of GCR activity. The basal medium RPMI 1640 with the addition of FBS was a satisfactory alternative for culturing the CHO-GCR cells. This study provided data that may contribute to the improvement of the GCR purification process. New research can be developed to improve the GCR purification process.

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.