Simultaneous isolation of proximal and distal lung progenitor cells from individual mice using a 3D printed guide reduces proximal cell contamination of distal lung epithelial cell isolations

Alsafadi, Hani N.; Stegmayr, John; Ptasinski, Victoria; Silva, Iran; Mittendorfer, Margareta; Murray, Lynne A.; Wagner, Darcy E.

doi:10.1016/j.stemcr.2022.11.002

Cited by 1 publication

(3 citation statements)

References 41 publications

(81 reference statements)

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“…In order to remove any potential bias due to changes in tissue thickness between biopsy pieces randomly allocated to the different fixatives, we first generated precision cut tissue slices using a vibratome (275 µm for brain and 200 µm for heart, liver, and kidney) and fixed parallel slices of each organ in one of the four commercially available fixatives overnight to mimic standard pathological fixation protocols. Next, we simultaneously processed all tissue pieces using a modification of a xylene-free tissue processing protocol we recently described for large animal organs which uses isopropanol instead of xylene for removal of ethanol to clear tissues and permit paraffin infiltration (Silva et al, 2022). We then created tissue specific tissue microarrays (TMA) containing one tissue slice fixed for each of the four different fixatives.…”

Section: Resultsmentioning

confidence: 99%

“…Additional details for all fixatives are listed in Table 1. The native biological tissue was processed as described previously using a xylene-free tissue processing routine (Silva et al, 2022). Prior to the embedding of the tissue, pieces were organized as "four spot" square tissue arrays into molten paraffin and allowed to cool as a single block.…”

Section: Tissue Harvest and Precision Tissue Slice Preparationmentioning

confidence: 99%

“…We recently described a xylene-free tissue processing protocol which substitutes isopropanol for xylene but was initially optimized for native biological (animal) tissue processing. We have additionally used this approach to histologically study cells seeded on polyester Transwell ™ membranes as well as Matrigel based organoids (Alsafadi et al, 2022;Silva et al, 2022). However, most of the previous literature and our own previous work used formalin-based crosslinking in conjunction with xylene-free tissue processing.…”

Section: Introductionmentioning

confidence: 99%

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Formalin-free fixation and xylene-free tissue processing preserves cell-hydrogel interactions for histological evaluation of 3D calcium alginate tissue engineered constructs

Silva

Gvazava

Wendi

et al. 2023

Front. Biomater. Sci.

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Histological evaluation of tissue-engineered products, including hydrogels for cellular encapsulation, is a critical and invaluable tool for assessing the product across multiple stages of its lifecycle from manufacture to implantation. However, many tissue-engineered products are comprised of polymers and hydrogels which are not optimized for use with conventional methods of tissue fixation and histological processing. Routine histology utilizes a combination of chemical fixatives, such as formaldehyde, and solvents such as xylene which have been optimized for use with native biological tissues due to their high protein and lipid content. Previous work has highlighted the challenges associated with processing hydrogels for routine histology due to their high water content and lack of diverse chemical moieties amenable for tissue fixation with traditional fixatives. Thus, hydrogel-based tissue engineering products are prone to histological artifacts during their validation which can lead to challenges in correctly interpreting results. In addition, chemicals used in conventional histological approaches are associated with significant health and environmental concerns due to their toxicity and there is thus an urgent need to identify suitable replacements. Here we use a multifactorial design of experiments approach to identify processing parameters capable of preserving cell-biomaterial interactions in a prototypical hydrogel system: ionically crosslinked calcium alginate. We identify a formalin free fixative which better retains cell-biomaterial interactions and calcium alginate hydrogel integrity as compared to the state-of-the-art formalin-based approaches. In addition, we demonstrate that this approach is compatible with a diversity of manufacturing techniques used to fabricate calcium alginate-based scaffolds for tissue engineering and cell therapy, including histological evaluation of cellular encapsulation in 3D tubes and thin tissue engineering scaffolds (∼50 μm). Furthermore, we show that formalin-free fixation can be used to retain cell-biomaterial interactions and hydrogel architecture in hybrid alginate-gelatin based scaffolds for use with histology and scanning electron microscopy. Taken together, these findings are a significant step forward towards improving histological evaluation of ionically crosslinked calcium alginate hydrogels and help make their validation less toxic, thus more environmentally friendly and sustainable.

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Section: Resultsmentioning

confidence: 99%

Section: Tissue Harvest and Precision Tissue Slice Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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