CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids

Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples.

Kefir as a Prevention of Arsenic-mediated Toxicity in Uterine Female Rats

Background: kefir is a fermented milk product that demonstrates numerous health benefits including antioxidant and immunomodulatory. Aim: to study the protective effect kefir on the expression of estrogen receptor alpha (ERα) in endometrial stromal cells and endometrial thickness on female rats that were exposed to arsenic. Methods: twenty-five female Wistar rats (Rattus norvegicus) were divided into five groups (CRL, As, T1, T2, T3). Control group (given a normal diet), As group (given the normal diet and exposed to arsenic trioxide 2 mg/kgBW/day). The T1; T2; T3 were exposed to arsenic trioxide 2 mg/kgBW/day and treated with different doses of kefir (1.25; 2.5; and 5 mL/kgBW/day, respectively) for 35 days. The rats of group As treated with arsenic trioxide only and group CRL served as control with normal feed in water. Cytological samples were taken after 35 days of treatment and examined every day to see the rat oestrus phase, and the proestrus phase of the oestrous cycle was chosen for termination. Uterine tissue fixed in 10% neutral buffered formalin for tissue preparation. ERα expression in endometrial stromal cells was analized using immunohistochemistry method, endometrial thickness was observed using histopathological methods. Results: significant reduction of ERα expression in endometrial stromal cells and endometrial thickness in female rats exposed to arsenic were observed in groups on treated rats (p ≤ 0.000; 0.009, respectively). Conclusion: the administration of kefir in female Wistar rats exposed to arsenic had shown significantly differences on ERα expressions and endometrial thickness. The smallest dose of kefir (1.25 mL/kgBW/day) could increase ERα expression and endometrial thickness in female Wistar rats with arsenic exposure. Therefore kefir has protective effect related to female reproductive system.

Descemet Membrane Endothelial Keratoplasty: NGS Metagenomics-Optimized Tissue Preparation and Surgical Technique. Doctoral Thesis

Title: Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium. Aim: To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method. Methods: Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16S and 18S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments. Results: In both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples. Conclusion: Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turnaround time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.

Descemet Membrane Endothelial Keratoplasty: NGS Metagenomics-Optimized Tissue Preparation and Surgical Technique. Summary of the Doctoral Thesis

Title: Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium. Aim: To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method. Methods: Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16S and 18S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments. Results: In both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples. Conclusion: Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turnaround time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.

Patient-Derived Explants as a Precision Medicine Patient-Proximal Testing Platform Informing Cancer Management

Precision medicine approaches that inform clinical management of individuals with cancer are progressively advancing. Patient-derived explants (PDEs) provide a patient-proximal ex vivo platform that can be used to assess sensitivity to standard of care (SOC) therapies and novel agents. PDEs have several advantages as a patient-proximal model compared to current preclinical models, as they maintain the phenotype and microenvironment of the individual tumor. However, the longevity of PDEs is not compatible with the timeframe required to incorporate candidate therapeutic options identified by whole exome sequencing (WES) of the patient’s tumor. This review investigates how PDE longevity varies across tumor streams and how this is influenced by tissue preparation. Improving longevity of PDEs will enable individualized therapeutics testing, and thus contribute to improving outcomes for people with cancer.

Kefir as a Prevention of Arsenic-mediated Toxicity in Uterine Female Rats

Background: kefir is a fermented milk product that demonstrates numerous health benefits including antioxidant and immunomodulatory. Aim: to study the protective effect kefir on the expression of estrogen receptor alpha (ERα) in endometrial stromal cells and endometrial thickness on female rats that were exposed to arsenic. Methods: twenty-five female Wistar rats (Rattus norvegicus) were divided into five groups (CRL, As, T1, T2, T3). Control group (given a normal diet), As group (given the normal diet and exposed to arsenic trioxide 2 mg/kgBW/day). The T1; T2; T3 were exposed to arsenic trioxide 2 mg/kgBW/day and treated with different doses of kefir (1.25; 2.5; and 5 mL/kgBW/day, respectively) for 35 days. The rats of group As treated with arsenic trioxide only and group CRL served as control with normal feed in water. Cytological samples were taken after 35 days of treatment and examined every day to see the rat oestrus phase, and the proestrus phase of the oestrous cycle was chosen for termination. Uterine tissue fixed in 10% neutral buffered formalin for tissue preparation. ERα expression in endometrial stromal cells was analized using immunohistochemistry method, endometrial thickness was observed using histopathological methods. Results: significant reduction of ERα expression in endometrial stromal cells and endometrial thickness in female rats exposed to arsenic were observed in groups on treated rats (p ≤ 0.000; 0.009, respectively). Conclusion: the administration of kefir in female Wistar rats exposed to arsenic had shown significantly differences on ERα expressions and endometrial thickness. The smallest dose of kefir (1.25 mL/kgBW/day) could increase ERα expression and endometrial thickness in female Wistar rats with arsenic exposure. Therefore kefir has protective effect related to female reproductive system.

Tissue Optimisation Strategies for High Quality Ex Vivo Diffusion Imaging

Ex vivo diffusion imaging can be used to study healthy and pathological tissue microstructure in the rodent brain with microscopic resolution, providing a link between in vivo MRI and ex vivo microscopy techniques. A major challenge for the successful acquisition of ex vivo diffusion imaging data however are changes in the relaxivity and diffusivity of brain tissue following perfusion fixation. In this study we address this question by examining the combined effects of tissue preparation factors that influence image quality, including tissue rehydration time, fixative concentration and contrast agent concentration. We present an optimisation strategy combining these factors to manipulate the T1 and T2 of fixed tissue and maximise signal-to-noise ratio (SNR) efficiency. Applying this strategy in the rat brain resulted in a doubling of SNR and an increase in SNR per unit time by 135% in grey matter and 88% in white matter. This enabled the acquisition of excellent quality high-resolution (78 μm isotropic voxel size) diffusion data in less than 4 days, with a b-value of 4000 s/mm2, 30 diffusion directions and a field of view of 40 x 13 x 18 mm, using a 9.4 Tesla scanner with a standard 39 mm volume coil and a 660 mT/m 114 mm gradient insert. It was also possible to achieve comparable data quality for a standard resolution (150 μm) diffusion dataset in 21/4 hours. In conclusion, the optimisation strategy presented here may be used to improve signal quality, increase spatial resolution and/or allow faster acquisitions in preclinical ex vivo diffusion MRI experiments.

Targeted ExSeq -- Tissue Preparation v2

This protocol accompanies Expansion Sequencing (ExSeq), and describes the tissue preparation for Targeted ExSeq. The steps described here are a generalization of the protocols used for figures 4-6 of the paper, and represent our recommendations for future users of the technology. Fig. 1 shows the structure of the protocol schematically. There are three possible tissue preparation routes described in this protocol that are applicable to different experimental systems. Option (A): harvesting tissue from model organisms that can be transcardially perfused with PFA, followed by sectioning using a vibratome. We typically use this workflow for work on mouse brain sections (see figures 4-5 of ExSeq paper). Option (B): transcardially perfusing with PFA, followed by cryoprotection and cryosectioning. We occasionally use this protocol for work on mouse brain sections. Option (C): snap-freezing fresh tissue (i.e., human tumor biopsy samples, or freshly harvested tissue from mice), followed by cryoprotection and cryosectioning (see figures 2 and 6 of ExSeq paper). The final result of options (A), (B), and (C) is the preparation of fixed tissue sections (either on a glass slide or free-floating). The protocols then briefly converge for optional antibody staining, treatment with LabelX, a chemical that enables anchoring of RNA to the expansion microscopy (ExM) hydrogel, followed by casting of the the ExM gel. There are minor differences in these steps between free-floating and slide-mounted tissue sections, which are noted in the individual steps. The next step, digestion, is tissue-type dependent and may require some optimization for your tissue type. We provide two potential options here: (1) a gentle digestion for tissues such as mouse brain, and (2) a harsh digestion for non-brain tissues such as tumor biopies. The protocols then converge again for the rest of the process. After digestion, the gels are expanded and re-embedded within a second non-expanding hydrogel to lock in the sample size. The carboxylates within the expansion gel are then chemically passivated, enabling enzymatic reactions to be performed within the gel. The samples are now ready for library preparation. In more detail: Steps 1-4 describe the preparation of reagents for downstream steps. The protocol begins either along options (A)/(B), the Transcardial PFA perfusion path (Step 5, continuing to vibratome sectioning in Steps 6-7 for option (A), or cryotome sectioning in Steps 9-10 for option (B)), or along option (C), the Fresh Frozen path (Step 8, continuing to cryotome sectioning in Steps 9-10). The protocols then converge for optional antibody staining (Step 11), followed by LabelX anchoring (Step 12), optional sample trimming (Step 13), and formation of the expansion microscopy gel (Step 14). The details of the digestion step are tissue-type dependent (Step 15). The protocol then concludes with expansion (Step 16), re-embedding (Step 17), passivation, and optional trimming (Steps 18-19). This protocol was used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP). The tissue for this work was collected (see HTAPP-specific tissue collection protocol). The tissue sections were then frozen, cryosectioned, post-fixed, and permeabilized (following steps 9-10). No antibody staining was performed (skipping optional step 11). The sections were then treated with LabelX and gelled (steps 12-14). The gels were then digested using the robust digestion option in steps 15-16. The samples were then re-embedded, passivated, and trimmed (following steps 17-19).

Targeted Expansion Sequencing Protocols v2

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

Targeted Expansion Sequencing Protocols v3

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).