Introdução à Cultura de Células

   Aula_1-Cultura de Células.pptx   

Aula 1

Glioblastoma multiforme (GBM), an extremely invasive and high-grade (grade IV) glioma, is the most common and aggressive form of brain cancer. It has a poor prognosis, with a median overall survival of only 11 months in the general GBM population and 14.6 to 21 months in clinical trial participants with standard GBM therapies, including maximum safe craniotomy, adjuvant radiation, and chemotherapies. Therefore, new approaches for developing efective treatments, such as a tool for assessing tumor cell drug response before drug treatments are administered, are urgently needed to improve patient survival. To address this issue, we developed an improved brain cancer chip with a difusion prevention mechanism that blocks drugs crossing from one channel to another. In the current study, we demonstrate that the chip has the ability to culture 3D spheroids from patient tumor specimen-derived GBM cells obtained from three GBM patients. Two clinical drugs used to treat GBM, temozolomide (TMZ) and bevacizumab (Avastin, BEV), were applied and a range of relative concentrations was generated by the microfuidic channels in the brain cancer chip. The results showed that TMZ works more efectively when used in combination with BEV compared to TMZ alone. We believe that this low-cost brain cancer chip could be further developed to generate optimal combination of chemotherapy drugs tailored to individual GBM patients.

  Seminário 1: Drug Screening of Human GBM Spheroids in Brain Cancer Chip   

  Seminário 1: Drug Screening of Human GBM Spheroids in Brain Cancer Chip   

Glioblastoma multiforme (GBM), an extremely invasive and high-grade (grade IV) glioma, is the most common and aggressive form of brain cancer. It has a poor prognosis, with a median overall survival of only 11 months in the general GBM population and 14.6 to 21 months in clinical trial participants with standard GBM therapies, including maximum safe craniotomy, adjuvant radiation, and chemotherapies. Therefore, new approaches for developing efective treatments, such as a tool for assessing tumor cell drug response before drug treatments are administered, are urgently needed to improve patient survival. To address this issue, we developed an improved brain cancer chip with a difusion prevention mechanism that blocks drugs crossing from one channel to another. In the current study, we demonstrate that the chip has the ability to culture 3D spheroids from patient tumor specimen-derived GBM cells obtained from three GBM patients. Two clinical drugs used to treat GBM, temozolomide (TMZ) and bevacizumab (Avastin, BEV), were applied and a range of relative concentrations was generated by the microfuidic channels in the brain cancer chip. The results showed that TMZ works more efectively when used in combination with BEV compared to TMZ alone. We believe that this low-cost brain cancer chip could be further developed to generate optimal combination of chemotherapy drugs tailored to individual GBM patients.

The methods used to study neuroendocrinology have been as diverse as the discoveries

to come out of the field. Maintaining live neurones outside of a body in vitro

was important from the beginning, building on methods that dated back to at least

the first decade of the 20th Century. Neurosecretion defines an essential foundation

of neuroendocrinology based on work that began in the 1920s and 1930s. Throughout

the first half of the 20th Century, many paradigms arose for studying everything

from single neurones to whole organs in vitro. Two of these survived as preeminent

systems for use throughout the second half of the century: cell cultures and explant

systems. Slice cultures and explants that emerged as organotypic technologies included

such neuroendocrine organs such as the brain, pituitary, adrenals and intestine.

The vast majority of these studies were carried out in static cultures for which

media were changed over a time scale of days. Tissues were used for experimental

techniques such as electrical recording of neuronal physiology in single cells and observation

by live microscopy. When maintained in vitro, many of these systems only

partially capture the in vivo physiology of the organ system of interest, often because

of a lack of cellular diversity (eg, neuronal cultures lacking glia). Modern microfluidic

methodologies show promise for organ systems, ranging from the reproductive

to the gastrointestinal to the brain. Moving forward and striving to understand the

mechanisms that drive neuroendocrine signalling centrally and peripherally, there

will always be a need to consider the heterogeneous cellular compositions of organs

in vivo.

  Seminário_From organotypic culture to body.pdf   

Background aims. Mesenchymal stromal cells (MSCs) are being investigated for use in cell therapy. The extensive in vitro expansion necessary to obtain sufficient cells for clinical use increases the risk that genetically abnormal cells will arise and be propagated during cell culture. Genetic abnormalities may lead to transformation and poor performance in clinical use, and are a critical safety concern for cell therapies using MSCs. Methods. We used spectral karyotyping (SKY) to investigate the genetic stability of human MSCs from ten donors during passaging. Results. Our data indicate that chromosomal abnormalities exist in MSCs at early passages and can be clonally propagated.The karyotypic abnormalities observed during our study diminished during passage. Conclusions. Karyotyping of MSCs reveals characteristics which may be valuable in deciding the suitability of cells for further use. Karyotypic analysis is useful for monitoring the genetic stability of MSCs during expansion

  Seminário_Chromosomal stability of mesenchymal stromal cells during.pdf   

Malignant bone disease (MBD) occurs when tumors establish in bone, causing catastrophic tissue damage as a result

of accelerated bone destruction and inhibition of repair. The resultant so-called osteolytic lesions (OL) take the form of

tumor-filled cavities in bone that cause pain, fractures, and associated morbidity. Furthermore, the OL

microenvironment can support survival of tumor cells and resistance to chemotherapy. Therefore, a deeper

understanding of OL formation and MBD progression is imperative for the development of future therapeutic

strategies. Herein, we describe a novel in vitro platform to study bone–tumor interactions based on three-dimensional

co-culture of osteogenically enhanced human mesenchymal stem cells (OEhMSCs) in a rotating wall vessel bioreactor

(RWV) while attached to micro-carrier beads coated with extracellular matrix (ECM) composed of factors found in

anabolic bone tissue. Osteoinhibition was recapitulated in this model by co-culturing the OEhMSCs with a

bone–tumor cell line (MOSJ-Dkk1) that secretes the canonical Wnt (cWnt) inhibitor Dkk-1, a tumor-borne

osteoinhibitory factor widely associated with several forms of MBD, or intact tumor fragments from Dkk-1 positive

patient-derived xenografts (PDX). Using the model, we observed that depending on the conditions of growth, tumor

cells can biochemically inhibit osteogenesis by disrupting cWnt activity in OEhMSCs, while simultaneously coengrafting

with OEhMSCs, displacing them from the niche, perturbing their activity, and promoting cell death. In the

absence of detectable co-engraftment with OEhMSCs, Dkk-1 positive PDX fragments had the capacity to enhance

OEhMSC proliferation while inhibiting their osteogenic differentiation. The model described has the capacity to

provide new and quantifiable insights into the multiple pathological mechanisms of MBD that are not readily

measured using monolayer culture or animal models.

  Seminário 2: Three-dimensional in vitro modeling of malignant bone disease recapitulates experimentally accessible mechanisms of osteoinhibition   

Stem cells (SCs) hold great promise for cell therapy, tissue engineering, and regenerative medicine as well

as pharmaceutical and biotechnological applications. They have the capacity to self-renew and the ability

to differentiate into specialized cell types depending upon their source of isolation. However, use of SCs for

clinical applications requires a high quality and quantity of cells. This necessitates large-scale expansion

of SCs followed by efficient and homogeneous differentiation into functional derivatives. Traditional

methods for maintenance and expansion of cells rely on two-dimensional (2-D) culturing techniques

using plastic culture plates and xenogenic media. These methods provide limited expansion and cells

tend to lose clonal and differentiation capacity upon long-term passaging. Recently, new approaches

for the expansion of SCs have emphasized three-dimensional (3-D) cell growth to mimic the in vivo

environment. This review provides a comprehensive compendium of recent advancements in culturing

SCs using 2-D and 3-D techniques involving spheroids, biomaterials, and bioreactors. In addition, potential

challenges to achieve billion-fold expansion of cells are discussed.

  Seminário_Advances and challenges in stem cell culture.pdf