BRPI1103059B1 - PROCESS FOR OBTAINING A CULTIVATION SUBSTRATE FOR PLURIPOTENT STEM CELLS AND CULTIVATION SUBSTRATE PRODUCED BY THE SAME - Google Patents
PROCESS FOR OBTAINING A CULTIVATION SUBSTRATE FOR PLURIPOTENT STEM CELLS AND CULTIVATION SUBSTRATE PRODUCED BY THE SAME Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
processo para a obtenção de um substrato de cultivo para células-tronco pluripotentes e substrato de cultivo produzido pelo mesmo. o presente pedido de patente de invenção compreende um substrato para o cultivo de células-tronco pluripotentes, o qual é gerado a partir da fixação por etanol de células de fibroblastos fetais de camundongo, e o processo de obtenção do mesmo. a invenção tem o objetivo de estabelecer um substrato com a complexidade necessária para garantir pelo menos 95% das células mantidas pluripotentes. além disso, o uso de camadas de células alimentadoras é substituído, as condições de cultivo sào mantidas e a contaminação com moléculas animais pela matriz é eliminada, sendo um substrato de baixo custo e compatível com a demanda científica nacional.process for obtaining a cultivation substrate for pluripotent stem cells and cultivation substrate produced therefrom. The present patent application comprises a substrate for the cultivation of pluripotent stem cells, which is generated from the ethanol fixation of mouse fetal fibroblast cells, and the process of obtaining the same. The invention aims to establish a substrate with the necessary complexity to guarantee at least 95% of cells maintained pluripotent. Furthermore, the use of feeder cell layers is replaced, cultivation conditions are maintained and contamination with animal molecules by the matrix is eliminated, making it a low-cost substrate compatible with national scientific demand.
Description
O presente pedido de patente descreve um substrato de cultivo para células-tronco embrionárias humanas e seu processo de obtenção, o qual se destina ao campo de produtos para pesquisa científica na área biológica e possui potencial para futuro uso na clínica médica.The present patent application describes a culture substrate for human embryonic stem cells and its obtaining process, which is intended for the field of products for scientific research in the biological area and has potential for future use in clinical medicine.
A invenção pode ser utilizada para todos os tipos de células pluripotentes (embrionárias e induzidas), tanto murinas quanto humanas. Também pode ser produzido em larga escala, gerando produtos de baixa perecibilidade.The invention can be used for all types of pluripotent cells (embryonic and induced), both murine and human. It can also be produced on a large scale, generating products with low perishability.
A manutenção das características das células-tronco pluripotentes demanda o uso de substratos complexos, tais como camadas de células alimentadoras de fontes animais ou extratos completos de matriz extracelular.Maintaining the characteristics of pluripotent stem cells requires the use of complex substrates, such as feeder cell layers from animal sources or complete extracts of extracellular matrix.
Tais substratos permitem o crescimento celular e sua manutenção funcional, porém, ao mesmo tempo, contaminam as células-tronco com moléculas imunogênicas que podem causar problemas em casos de transplantes.Such substrates allow cell growth and its functional maintenance, however, at the same time, they contaminate stem cells with immunogenic molecules that can cause problems in cases of transplantation.
As principais formas de produzir substratos de cultivo para células-tronco embrionárias são a matriz produzida in loco, matrigel e proteínas purificadas. A matriz produzida in loco e o matrigel consistem em substratos completos de matriz extracelular, enquanto as proteínas purificadas representam um substrato específico, que contém apenas um ou dois tipos de moléculas presentes.The main ways to produce culture substrates for embryonic stem cells are the matrix produced in loco, matrigel and purified proteins. The matrix produced in loco and the matrigel consist of complete extracellular matrix substrates, while the purified proteins represent a specific substrate, which contains only one or two types of molecules present.
A matriz produzida in loco consiste no cultivo de células produtoras de matriz que, após produção da matriz, são lisadas e eliminadas, deixando o substrato livre de células vivas. Uma das maneiras mais comuns de realização deste procedimento é pelo tratamento químico com a amónia, cuja função é eliminar o corpo celular e deixar o substrato apenas com as moléculas da matriz. No entanto, as moléculas não são fixadas ao substrato e são perecíveis, apresentando um curto período de uso. Para o cultivo específico de células-tronco pluripotentes humanas, tal substrato só pode ser utilizado em combinação com outros fatores.The matrix produced in loco consists of the cultivation of matrix-producing cells which, after matrix production, are lysed and eliminated, leaving the substrate free of live cells. One of the most common ways of performing this procedure is by chemical treatment with ammonia, whose function is to eliminate the cell body and leave the substrate with only the matrix molecules. However, the molecules are not fixed to the substrate and are perishable, with a short period of use. For the specific culture of human pluripotent stem cells, this substrate can only be used in combination with other factors.
O extrato de matriz em forma de gel é comercializado sob o nome de matrigel e consiste em uma mistura gelatinosa de proteínas, lipídios e carboidratos, que são produzidos por células de camundongo. O matrigel tem sido utilizado no cultivo de células-tronco embrionárias humanas há cerca de uma década, porém também requer o uso combinado com outros fatores, o que o torna mais custoso.The matrix extract in gel form is marketed under the name matrigel and consists of a gelatinous mixture of proteins, lipids and carbohydrates, which are produced by mouse cells. Matrigel has been used in the cultivation of human embryonic stem cells for about a decade, but it also requires its use in combination with other factors, which makes it more costly.
O uso de proteínas purificadas de matriz extracelular, como a laminina e a fibronectina, também requer condições especiais de cultivo, uma vez que o substrato acelular produzido por proteínas purificadas não é tão rico quanto a matriz completa produzida pelo próprio fibroblasto.The use of purified extracellular matrix proteins, such as laminin and fibronectin, also requires special cultivation conditions, since the acellular substrate produced by purified proteins is not as rich as the complete matrix produced by the fibroblast itself.
Células-tronco pluripotentes humanas são cultivadas em meio condicionado, os quais podem ser: - Substrato celular (MEF - fibroblastos embrionários murinos) + FGF-2 (4 a 8ng/mL) - Substrato acelular + FGF-2 (40 a 100ng/mL) - Substrato acelular + FGF-2 (4 a 8ng/mL) + meio condicionado com MEFHuman pluripotent stem cells are cultivated in conditioned medium, which can be: - Cellular substrate (MEF - murine embryonic fibroblasts) + FGF-2 (4 to 8ng/ml) - Acellular substrate + FGF-2 (40 to 100ng/ml) ) - Acellular substrate + FGF-2 (4 to 8ng/ml) + MEF conditioned medium
Assim, pode-se observar que, quando o substrato de cultivo é acelular, não há modelo de cultivo de células- tronco em baixa concentração de FGF-2 sem que haja o condicionamento do meio com MEF,Thus, it can be observed that, when the culture substrate is acellular, there is no model of stem cell culture in low concentration of FGF-2 without conditioning the medium with MEF,
A presente invenção descreve um substrato no qual as camadas de células alimentadoras são substituídas por células de fibroblastos fetais de camundongo. Assim, obtém- se um substrato altamente eficiente, que apresenta baixo teor de contaminação cruzada, armazenamento simples e por longo período de tempo sem alteração das características, além de baixo custo de produção.The present invention describes a substrate in which feeder cell layers are replaced by mouse fetal fibroblast cells. Thus, a highly efficient substrate is obtained, which has a low cross-contamination content, simple storage and for a long period of time without changing its characteristics, in addition to a low production cost.
O documento de anterioridade US 2003175956 Al descreve um método que permite a proliferação indiferenciada de células-tronco em uma matriz extracelular fibroblástica com meio nutriente adicionado de FGF, no qual o ambiente de crescimento não apresenta células alimentadoras. A matriz descrita neste documento só é eficiente se for utilizado um meio condicionado na presença de FGF-2 (5 ng/mL), enquanto que, no presente pedido, a matriz pode ser utilizada com qualquer meio de cultivo próprio para células-tronco pluripotentes humanas. Além disso, o modo de preparo do substrato também é diferente, tratando-se de uma lise celular provocada pelo hidróxido de amónio.Priority document US 2003175956 A1 describes a method that allows the undifferentiated proliferation of stem cells in a fibroblastic extracellular matrix with nutrient medium added with FGF, in which the growth environment does not present feeder cells. The matrix described in this document is only efficient if a conditioned medium is used in the presence of FGF-2 (5 ng/ml), whereas, in the present application, the matrix can be used with any culture medium suitable for pluripotent stem cells human beings. Furthermore, the way in which the substrate is prepared is also different, since it is a cell lysis caused by ammonium hydroxide.
O documento de anterioridade CN 1986781 A revela um método para o cultivo de células-tronco mesenquimais em um ambiente contendo uma matriz extracelular separado a partir de fibroblasto humano. Por outro lado, a invenção pleiteada neste pedido destina-se ao cultivo de células-tronco pluripotentes.Priority document CN 1986781 A discloses a method for culturing mesenchymal stem cells in an environment containing an extracellular matrix separated from human fibroblasts. On the other hand, the invention claimed in this application is intended for the cultivation of pluripotent stem cells.
O documento europeu EP 2267116 Al se refere a um meio nutriente, ausente de células alimentadoras e com pelo menos 80 ng/mL de FGF, para a proliferação indiferenciada de células-tronco pluripotentes. De acordo com o protocolo apresentado na EP, a adaptação celular na matriz ocorre após 6 passagens (aproximadamente 42 dias), enquanto que, na presente invenção, apenas 2 passagens (cerca de 14 dias) são suficientes para a adaptação. Além disso, o substrato acelular ora descrito é composto de uma matriz completa gerada por fibroblastos.European document EP 2267116 A1 refers to a nutrient medium, absent from feeder cells and with at least 80 ng/ml of FGF, for the undifferentiated proliferation of pluripotent stem cells. According to the protocol presented in the EP, cell adaptation to the matrix occurs after 6 passages (approximately 42 days), whereas, in the present invention, only 2 passages (approximately 14 days) are sufficient for adaptation. Furthermore, the acellular substrate just described is composed of a complete matrix generated by fibroblasts.
O documento coreano KR 20080087264 A revela uma matriz de fibroblasto de placenta para proliferação e aderência de células-tronco mesenquimais oriundas do cordão umbilical, enquanto que a presente invenção serve para o cultivo de células-tronco pluripotentes e a preparação do substrato é diferente.Korean document KR 20080087264 A discloses a placental fibroblast matrix for proliferation and adherence of mesenchymal stem cells from the umbilical cord, while the present invention serves for the cultivation of pluripotent stem cells and the substrate preparation is different.
O documento WO 2009079409 Al descreve um substrato de cultivo que compreende uma mistura de fibronectina e albumina (proteínas purificadas), o qual deve ser acrescido de altas concentrações de FGF-2 com a finalidade de manter as características das células-tronco.The document WO 2009079409 Al describes a cultivation substrate that comprises a mixture of fibronectin and albumin (purified proteins), which must be added with high concentrations of FGF-2 in order to maintain the characteristics of the stem cells.
O pedido brasileiro BR 0514778 A ensina um método para o cultivo de células-tronco humanas não diferenciadas em matriz enriquecida na ausência de células alimentadoras. As células-tronco são cultivadas em alta concentração de FGF-2 (40 a 100 ng/mL), enquanto que na presente invenção utiliza-se apenas 4 a 8 ng/mL de FGF-2.Brazilian application BR 0514778 A teaches a method for cultivating undifferentiated human stem cells in enriched matrix in the absence of feeder cells. Stem cells are cultivated in a high concentration of FGF-2 (40 to 100 ng/ml), whereas in the present invention only 4 to 8 ng/ml of FGF-2 is used.
O pedido BR 0514641 A ensina a formação de uma matriz humanizada para cultura de células-tronco embrionárias compreendendo colágeno, fibronectina, vitronectina e laminina, com adição de fator de crescimento de fibroblasto em uma concentração de pelo menos 40 ng/ml. No entanto, uma mistura de proteínas como descrito neste pedido não possui as mesmas características biológicas de uma matriz completa.Application BR 0514641 A teaches the formation of a humanized matrix for culturing embryonic stem cells comprising collagen, fibronectin, vitronectin and laminin, with the addition of fibroblast growth factor at a concentration of at least 40 ng/ml. However, a protein mixture as described in this application does not have the same biological characteristics as a complete matrix.
O presente pedido de patente de invenção compreende um substrato para o cultivo de células-tronco pluripotentes, o qual é gerado a partir da fixação alcoólica de células de fibroblastos fetais de camundongo, e o processo de obtenção do mesmo.The present patent application comprises a substrate for the cultivation of pluripotent stem cells, which is generated from the alcoholic fixation of mouse fetal fibroblast cells, and the process for obtaining it.
A invenção tem o objetivo de estabelecer um substrato com a complexidade necessária para garantir pelo menos 95% das células mantidas pluripotentes.The invention aims to establish a substrate with the necessary complexity to guarantee at least 95% of cells kept pluripotent.
Além disso, o uso de camadas de células alimentadoras é substituído, as condições de cultivo são mantidas e a contaminação com moléculas animais advindas da matriz é eliminada, sendo um substrato de baixo custo e compatível com a demanda científica nacional.In addition, the use of feeder cell layers is replaced, cultivation conditions are maintained and contamination with animal molecules from the matrix is eliminated, making it a low-cost substrate that is compatible with national scientific demand.
As Figuras IA e 1B são fotomicrografias que mostram a morfologia típica de colônias características de células- tronco pluripotentes sob condição controle (A) e MEF etanol (B), respectivamente.Figures IA and 1B are photomicrographs showing the typical morphology of characteristic pluripotent stem cell colonies under control (A) and MEF ethanol (B), respectively.
A Figura 1C é um gráfico que demonstra o ritmo de crescimento celular em ambas as condições de (A) e (B).Figure 1C is a graph demonstrating the cell growth rate under both the conditions of (A) and (B).
As Figuras 2A, 2B, 2C e 2D são gráficos que mostram os níveis dos marcadores de pluripotência Oct-4, SOX-2, SSEA-4 e TRA-1-60, respectivamente, nas células-tronco cultivadas sob condição controle e MEF etanol.Figures 2A, 2B, 2C and 2D are graphs showing the levels of pluripotency markers Oct-4, SOX-2, SSEA-4 and TRA-1-60, respectively, in stem cells grown under control and MEF ethanol conditions .
As Figuras 3A e 3B são fotomicrografias que mostram a morfologia típica de corpos embrióides formados a partir de células-tronco cultivadas sob os substratos controle (A) e etanol (B), respectivamente.Figures 3A and 3B are photomicrographs showing the typical morphology of embryoid bodies formed from stem cells cultured under control (A) and ethanol (B) substrates, respectively.
A Figura 3C é um gráfico que analisa o diâmetro dos corpos embrióides em ambas as condições de (A) e (B).Figure 3C is a graph that analyzes the diameter of embryoid bodies under both conditions (A) and (B).
A Figura 4 é um gráfico que demonstra a quantificação dos níveis de ácido siálico Neu5Gc nas células-tronco.Figure 4 is a graph demonstrating the quantification of sialic acid Neu5Gc levels in stem cells.
O presente pedido de patente descreve um substrato de cultivo para células-tronco embrionárias humanas, o qual é produzido pelo tratamento de fibroblastos de camundongo com etanol, que promove a lise celular e leva à fixação da matriz.The present patent application describes a culture substrate for human embryonic stem cells, which is produced by treating mouse fibroblasts with ethanol, which promotes cell lysis and leads to matrix fixation.
O substrato ora descrito apresenta uma complexidade mínima necessária para garantir pelo menos 95% das células mantidas pluripotentes, além de apresentar baixo custo e ser imunocompatível.The substrate described herein has the minimum complexity necessary to guarantee at least 95% of cells kept pluripotent, in addition to being low cost and immunocompatible.
O substrato desta invenção é obtido da seguinte forma:The substrate of this invention is obtained as follows:
As placas de petri próprias para o cultivo celular (ou seja, que apresentam superfície negativamente carregada) são rinsadas com uma solução 0,1% de gelatina estéril e colocadas abertas em fluxo laminar até a secagem do líquido.Petri dishes suitable for cell culture (ie, which have a negatively charged surface) are rinsed with a 0.1% sterile gelatin solution and placed open in a laminar flow until the liquid has dried.
Este passo tem a função de garantir a boa adesão das células a serem cultivadas à superfície da placa.This step has the function of guaranteeing the good adhesion of the cells to be cultured to the surface of the plate.
Prepara-se o meio de cultivo dos fibroblastos, o qual consiste em: - DMEM/F12 (meio base) - 10% soro fetal bovino - 2mM glutamina - 0,2% 2-mercaptoetanolThe fibroblast culture medium is prepared, which consists of: - DMEM/F12 (base medium) - 10% fetal bovine serum - 2mM glutamine - 0.2% 2-mercaptoethanol
O criotubo contendo os fibroblastos embrionários murinos é descongelado em banho-maria a 37 °C com leve agitação manual por 1 a 2 minutos e, em seguida, são diluídas em meio de fibroblasto na proporção de 1 parte de meio de congelamento para 2 partes de meio de fibroblasto.The cryotube containing the murine embryonic fibroblasts is thawed in a water bath at 37 °C with gentle manual shaking for 1 to 2 minutes and then diluted in fibroblast medium in the proportion of 1 part of freezing medium to 2 parts of fibroblast medium.
O material é centrifugado a 240 g por 5 minutos, o sobrenadante sendo descartado e o pellet (células) sendo suspenso novamente em meio de fibroblastos.The material is centrifuged at 240 g for 5 minutes, the supernatant being discarded and the pellet (cells) being resuspended in fibroblast medium.
Os fibroblastos descongelados são plaqueados nas placas em uma densidade de 2,6x104 células/cm2 e incubados a 37°C em 5% CO2 por 4 8 horas.Thawed fibroblasts are plated onto the plates at a density of 2.6x104 cells/cm2 and incubated at 37°C in 5% CO2 for 48 hours.
Após 48 horas de incubação, as placas cultivadas são rinsadas com água estéril, com volume suficiente para cobrir toda a superfície da placa.After 48 hours of incubation, the cultured plates are rinsed with sterile water, with sufficient volume to cover the entire surface of the plate.
A água é aspirada e aplica-se o mesmo volume de etanol 70%, deixando-se a placa tampada no fluxo laminar por 5 minutos ã temperatura ambiente, sendo o etanol aspirado em seguida.The water is aspirated and the same volume of 70% ethanol is applied, leaving the covered plate in the laminar flow for 5 minutes at room temperature, and the ethanol is then aspirated.
A placa é novamente rinsada com etanol 70%, aspirada e seca destampada em fluxo laminar por aproximadamente 5 a 10 minutos.The plate is again rinsed with 70% ethanol, aspirated and dried uncapped in a laminar flow for approximately 5 to 10 minutes.
A placa é selada com parafilm ou guardada em embalagem estéril e acondicionada a 4 °C por até 2 meses ou a temperatura ambiente por até 1 semana.The plate is sealed with parafilm or stored in sterile packaging and stored at 4 °C for up to 2 months or at room temperature for up to 1 week.
Ainda, para armazenamentos mais longos (superior a 2 meses), a placa deve ser acondicionada com solução de etanol 70% a -20°C.Also, for longer storage (greater than 2 months), the plate must be conditioned with a 70% ethanol solution at -20°C.
Para utilização da placa para o cultivo de células- tronco pluripotentes, deve-se deixar a placa atingir a temperatura ambiente e plaqueá-la.To use the plate for the cultivation of pluripotent stem cells, the plate should be allowed to reach room temperature and plated.
Em caso de células-tronco embrionárias murinas, o meio deve ser complementado com o fator LIF (mesma concentração utilizada para o cultivo sobre camadas alimentadoras inativadas) .In the case of murine embryonic stem cells, the medium must be supplemented with the LIF factor (the same concentration used for cultivation on inactivated feeder layers) .
Para células-tronco embrionárias humanas, o uso do fator FGF-2 na concentração de 8 ng/mL é suficiente para a manutenção das características das células, mas pode-se utilizar concentrações superiores de acordo com o meio de cultivo utilizado.For human embryonic stem cells, the use of FGF-2 factor at a concentration of 8 ng/mL is sufficient to maintain the characteristics of the cells, but higher concentrations can be used depending on the culture medium used.
As figuras IA e 1B representam fotomicrografias em campo claro (escala 50μm) que mostram a morfologia típica de colônia característica das células-tronco pluripotentes.Figures IA and 1B represent brightfield photomicrographs (50μm scale) that show the typical colony morphology characteristic of pluripotent stem cells.
Células da linhagem H9 foram cultivadas sobre uma condição controle (A) e no substrato MEF etanol (B) aqui descrito.Cells from the H9 lineage were cultured under a control condition (A) and in the ethanol MEF substrate (B) described here.
Conforme pode ser observado no gráfico 1C, o ritmo de crescimento das células H9 não foi alterado ao se comparar as condições controle e MEF etanol.As can be seen in graph 1C, the growth rate of H9 cells was not altered when comparing the control and MEF ethanol conditions.
Assim, comprova-se que as células-tronco pluripotentes humanas mantêm sua morfologia e ritmo de crescimento característicos no substrato MEF etanol.Thus, it is proven that human pluripotent stem cells maintain their characteristic morphology and growth rate in the MEF ethanol substrate.
A figura 2 ilustra graficamente a quantificação dos marcadores de pluripotência Oct-4 (A) e SOX-2 (B), através de fotomicrografias de imunomarcação das células-tronco pluripotentes, e a quantificação dos marcadores SSEA-4 (C) e TRA-1-60 (D) através de análise por citometria.Figure 2 graphically illustrates the quantification of the pluripotency markers Oct-4 (A) and SOX-2 (B), through photomicrographs of immunostaining of pluripotent stem cells, and the quantification of the markers SSEA-4 (C) and TRA- 1-60 (D) by cytometric analysis.
Isso significa que as células-tronco pluripotentes humanas da linhagem H9 mantêm altos níveis dos marcadores de pluripotência Oct-4, SOX-2, SSEA-4 e TRA-1-60 quando cultivadas por pelo menos 100 dias nas condições controle e sobre o substrato MEF etanol.This means that human pluripotent stem cells of the H9 lineage maintain high levels of the pluripotency markers Oct-4, SOX-2, SSEA-4 and TRA-1-60 when cultured for at least 100 days under control conditions and on substrate Ethanol MEF.
As figuras 3A e 3B representam fotomicrografias em campo claro (escala lOOμm) de corpos embrióides formados a partir de células H9 cultivadas sobre uma condição controle (A) e no substrato MEF etanol (B) aqui descrito.Figures 3A and 3B represent brightfield photomicrographs (100μm scale) of embryoid bodies formed from H9 cells cultured under a control condition (A) and on the MEF ethanol substrate (B) described here.
Conforme pode ser observado no gráfico 3C, o diâmetro dos corpos embrióides formados não foi alterado ao se comparar as condições controle e MEF etanol.As can be seen in the 3C graph, the diameter of the embryoid bodies formed was not changed when comparing the control and MEF ethanol conditions.
Assim, comprova-se que as células-tronco pluripotentes humanas mantêm sua capacidade de diferenciação em corpos embrióides quando cultivadas no substrato MEF etanol.Thus, it is proven that human pluripotent stem cells maintain their ability to differentiate into embryoid bodies when cultivated in the MEF ethanol substrate.
A figura 4 ilustra graficamente a quantificação de imunomarcação do ácido siálico Neu5Gc por citometria das células-tronco pluripotentes.Figure 4 graphically illustrates the quantification of sialic acid Neu5Gc immunostaining by pluripotent stem cell cytometry.
Conforme pode ser observado, as células H9 cultivadas sobre a condições controle apresentam mais células positivas (38%) para NeuSGc do que as células H9 cultivadas sobre o substrato MEF etanol (29%).As can be seen, H9 cells grown on control conditions have more cells (38%) positive for NeuSGc than H9 cells grown on the substrate MEF ethanol (29%).
Além disso, fibroblastos embrionários murinos (MEF) foram utilizados como controle positivo do experimento e apresentaram 64% de células positivas.In addition, murine embryonic fibroblasts (MEF) were used as a positive control in the experiment and showed 64% of positive cells.
Isso significa que as células-tronco pluripotentes humanas da linhagem H9 mantidas sobre o substrato MEF 10 etanol apresentaram menores níveis do ácido siálico não- humano NeuSGc.This means that human pluripotent stem cells of the H9 lineage maintained on the substrate MEF 10 ethanol had lower levels of the non-human sialic acid NeuSGc.
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