An organoid is a 3D organization of cells that can recapitulate some of the structure and function of native tissue. Recent work has seen organoids gain prominence as a valuable model for studying ...tissue development, drug discovery, and potential clinical applications. The requirements for the successful culture of organoids in vitro differ significantly from those of traditional monolayer cell cultures. The generation and maturation of high-fidelity organoids entails developing and optimizing environmental conditions to provide the optimal cues for growth and 3D maturation, such as oxygenation, mechanical and fluidic activation, nutrition gradients, etc. To this end, we discuss the four main categories of bioreactors used for organoid culture: stirred bioreactors (SBR), microfluidic bioreactors (MFB), rotating wall vessels (RWV), and electrically stimulating (ES) bioreactors. We aim to lay out the state-of-the-art of both commercial and in-house developed bioreactor systems, their benefits to the culture of organoids derived from various cells and tissues, and the limitations of bioreactor technology, including sterilization, accessibility, and suitability and ease of use for long-term culture. Finally, we discuss future directions for improvements to existing bioreactor technology and how they may be used to enhance organoid culture for specific applications.
Biomarkers for α‐synuclein are needed for diagnosis and prognosis in Parkinson's disease (PD). Endogenous auto‐antibodies to α‐synuclein could serve as biomarkers for underlying synucleinopathy, but ...previous assessments of auto‐antibodies have shown variability and inconsistent clinical correlations. We hypothesized that auto‐antibodies to α‐synuclein could be diagnostic for PD and explain its clinical heterogeneity. To test this hypothesis, we developed an enzyme‐linked immunosorbent assay for measuring α‐synuclein auto‐antibodies in human samples. We evaluated 69 serum samples (16 healthy controls (HC) and 53 PD patients) and 145 CSF samples (52 HC and 93 PD patients) from our Institution. Both serum and CSF were available for 24 participants. Males had higher auto‐antibody levels than females in both fluids. CSF auto‐antibody levels were significantly higher in PD patients as compared with HC, whereas serum levels were not significantly different. CSF auto‐antibody levels did not associate with amyloid‐β1–42, total tau, or phosphorylated tau. CSF auto‐antibody levels correlated with performance on the Montreal Cognitive Assessment, even when controlled for CSF amyloidβ1–42. CSF hemoglobin levels, as a proxy for contamination of CSF by blood during lumbar puncture, did not influence these observations. Using recombinant α‐synuclein with N‐ and C‐terminal truncations, we found that CSF auto‐antibodies target amino acids 100 through 120 of α‐synuclein. We conclude that endogenous CSF auto‐antibodies are significantly higher in PD patients as compared with HC, suggesting that they could indicate the presence of underlying synucleinopathy. These auto‐antibodies associate with poor cognition, independently of CSF amyloidβ1–42, and target a select C‐terminal region of α‐synuclein.
Read the Editorial Highlight for this article on page 433.
Endogenous auto‐antibodies to α‐synuclein could be useful biomarkers in Parkinson's disease (PD), but previous attempts to measure auto‐antibodies have been inconclusive. We developed a reliable method to measure α‐synuclein auto‐antibodies in biofluid samples. We found significantly higher levels of auto‐antibodies in the cerebrospinal fluid (CSF) of PD patients than in healthy controls. In the serum, levels of auto‐antibodies were similar in both groups. Participants with higher levels of CSF auto‐antibodies tended to have worse cognitive performance, as measured by the Montreal Cognitive Assessment. These auto‐antibodies recognized the C‐terminal domain in α‐synuclein. This study suggests that auto‐antibodies to α‐synuclein could be useful biomarkers for diagnosis in PD and may correlate with clinical disease features.
Read the Editorial Highlight for this article on page 433.
Three-dimensional (3D) printing has the potential to be used for rapid-prototyping and inexpensive fabrication of bioreactors for advanced cell and tissue culture. However, the suitability of ...materials used for 3D printing these bioreactors that will be in direct contact with cells and culture media remains to be established. Many of the most common low-cost materials have not been thoroughly tested under stringent cell culture conditions, especially not with highly sensitive human cell types, such as induced pluripotent stem cells (hiPSCs). This study aims to characterize some 3D printed plastics, such as thermoplastics and photopolymers, focusing on the toxicity/cytocompatibility of the materials as assessed by hiPSC viability, retention of pluripotency, and cardiogenic differentiation potential. Experiments were conducted in a manner that simulates contact between 3D printed plastics and cell culture media, as found in a 3D printed bioreactor. Both photopolymers tested here reduced the viability of hiPSCs, but not of primary human fibroblasts, highlighting the importance of carrying out these tests with the cells of interest. The thermoplastics did not adversely affect stem cell viability, pluripotency, or cardiac differentiation potential. However, except for Nylon12, all thermoplastics deformed during autoclaving, leading us to choose Nylon12 as the most suitable material for bioreactor fabrication. This study represents a step forward in the use of 3D printing for the rapid, low-cost fabrication of custom-designed bioreactors.
•Photoresins are cytotoxic to human induced pluripotent stem cells (hiPSCs), but not to primary human dermal fibroblasts.•The effects of 3D printed plastic on hiPSC viability, pluripotency, and cardiac differentiation were assessed.•While all thermoplastics tested here on non-cytotoxic to hiPSCs, only Nylon 12 could withstand autoclave sterilization.
A contemporary pathology of science Modonesi, Carlo; Farina, Lorenzo; Licata, Ignazio ...
Annali dell'Istituto superiore di sanità,
2008, Letnik:
44, Številka:
3
Journal Article
Recenzirano
A contemporary pathology of science is outlined. This pathology suggests that "previous knowledge" drastically limits innovative thinking in science. In very raw "Bayesian" terms it is affirmed that ...a too rich and flexible a priori knowledge is detrimental to the appreciation of novelty coming from experimental results by both lowering the relative weight assigned to a posteriori contrasting evidence and adapting potentially revolutionary findings to an already existing frame.
Chimeric antigen receptor-modified T cell (CAR-T) technology, a promising immunotherapeutic tool, has not been applied specifically to treat liver metastases (LM). While CAR-T delivery to LM can be ...optimized by regional intrahepatic infusion, we propose that liver CD11b+Gr-1+ myeloid-derived suppressor cells (L-MDSC) will inhibit the efficacy of CAR-T in the intrahepatic space. We studied anti-CEA CAR-T in a murine model of CEA+ LM and identified mechanisms through which L-MDSC expand and inhibit CAR-T function. We established CEA+ LM in mice and studied purified L-MDSC and responses to treatment with intrahepatic anti-CEA CAR-T infusions. L-MDSC expanded threefold in response to LM, and their expansion was dependent on GM-CSF, which was produced by tumor cells. L-MDSC utilized PD-L1 to suppress anti-tumor responses through engagement of PD-1 on CAR-T. GM-CSF, in cooperation with STAT3, promoted L-MDSC PD-L1 expression. CAR-T efficacy was rescued when mice received CAR-T in combination with MDSC depletion, GM-CSF neutralization to prevent MDSC expansion, or PD-L1 blockade. As L-MDSC suppressed anti-CEA CAR-T, infusion of anti-CEA CAR-T in tandem with agents targeting L-MDSC is a rational strategy for future clinical trials.
Summary
Increased expression of SIRT1 extends the lifespan of lower organisms and delays the onset of age‐related diseases in mammals. Here, we show that SRT2104, a synthetic small molecule activator ...of SIRT1, extends both mean and maximal lifespan of mice fed a standard diet. This is accompanied by improvements in health, including enhanced motor coordination, performance, bone mineral density, and insulin sensitivity associated with higher mitochondrial content and decreased inflammation. Short‐term SRT2104 treatment preserves bone and muscle mass in an experimental model of atrophy. These results demonstrate it is possible to design a small molecule that can slow aging and delay multiple age‐related diseases in mammals, supporting the therapeutic potential of SIRT1 activators in humans.
Intrahepatic inflammation and liver T cell dysfunction due to obstructive jaundice are both ameliorated through abrogation of PD‐1 expression.
Biliary obstruction is a common clinical problem that is ...associated with intrahepatic inflammation and impaired immunity. PD‐1 is well known to mediate T cell dysfunction but has been reported to promote and attenuate acute inflammation in various injury models. With the use of a well‐established murine model of BDL, we studied the effects of intrahepatic PD‐1 expression on LTC function, inflammation, and cholestasis. Following BDL, PD‐1 expression increased significantly among LTCs. Increased PD‐1 expression following BDL was associated with decreased LTC proliferation and less IFN‐γ production. Elimination of PD‐1 expression resulted in significantly improved proliferative capacity among LTC following BDL, in addition to a more immunostimulatory cytokine profile. Not only was LTC function rescued in PD‐1−/− mice, but also, the degrees of biliary cell injury, cholestasis, and inflammation were diminished significantly compared with WT animals following BDL. PD‐1‐mediated acute inflammation following BDL was associated with expansions of intrahepatic neutrophil and Th17 cell populations, with the latter dependent on IL‐6. PD‐1 blockade represents an attractive strategy for reversing intrahepatic immunosuppression while limiting inflammatory liver damage.
Our phase I Hepatic Immunotherapy for Metastases (HITM) trial tested the safety of chimeric antigen receptor-modified T-cell (CAR-T) hepatic artery infusions (HAI) for unresectable carcinoembryonic ...antigen (CEA)+ liver metastases (LM). High neutrophil:lymphocyte ratios (NLR) predict poor outcome in cancer patients and we hypothesized that NLR changes would correlate with early responses to CAR-T HAI. Six patients completed the protocol. Three patients received CAR-T HAI in dose escalation (1 × 10(8), 1 × 10(9) and 1 × 10(10) cells) and the remainder received three doses (1 × 10(10) cells) with interleukin (IL)2 support. Serum cytokines and NLR were measured at multiple time points. The mean NLR for all patients was 13.9 (range 4.8-38.1). NLR increased in four patients following treatment with a mean fold change of 1.9. Serum IL6 levels and NLR fold changes demonstrated a trend towards a positive correlation (r=0.77, P=0.10). Patients with poor CEA responses were significantly more likely to have higher NLR level increases (P=0.048). Increased NLR levels were associated with poor responses following CAR-T HAI. NLR variations and associated cytokine changes may be useful surrogates of response to CAR-T HAI.
Imatinib mesylate is an effective treatment for metastatic gastrointestinal stromal tumor (GIST). However, most patients eventually develop resistance and there are few other treatment options. ...Immunotherapy using genetically modified or designer T cells (dTc) has gained increased attention for several malignancies in recent years. The aims of this study were to develop and test novel anti-KIT dTc engineered to target GIST cells.
Human anti-KIT dTc were created by retroviral transduction with novel chimeric immune receptors (CIR). The gene for stem cell factor (SCF), the natural ligand for KIT, was cloned into 1st generation (SCF-CD3ζ, 1st gen) and 2nd generation (SCF-CD28-CD3ζ, 2nd gen) CIR constructs. In vitro dTc proliferation and tumoricidal capacity in the presence of KIT+ tumor cells were measured. In vivo assessment of dTc anti-tumor efficacy was performed by treating immunodeficient mice harboring subcutaneous GIST xenografts with dTc tail vein infusions.
We successfully produced the 1st and 2nd gen anti-KIT CIR and transduced murine and human T cells. Average transduction efficiencies for human 1st and 2nd gen dTc were 50% and 42%. When co-cultured with KIT+ tumor cells, both 1st and 2nd gen dTc proliferated and produced IFNγ. Human anti-KIT dTc were efficient at lysing GIST in vitro compared to untransduced T cells. In mice with established GIST xenografts, treatment with either 1st or 2nd gen human anti-KIT dTc led to significant reductions in tumor growth rates.
We have constructed a novel anti-KIT CIR for production of dTc that possess specific activity against KIT+ GIST in vitro and in vivo. Further studies are warranted to evaluate the therapeutic potential and safety of anti-KIT dTc.
PDF
