Noninvasive Intravascular Microtransfusion in Colonial Tunicates

Abstract

Tunicates are a diverse group of worldwide marine filter-feeders that are vertebrates’ closest invertebrate relatives. Colonial tunicates are the only know chordates that have been shown to undergo whole-body regeneration (WBR). Botrylloides in particular can regenerate one fully functional adult from a minute fragment of their vascular system in as little as 10 days. This regenerative process relies on the proliferation of circulating stem cells, likely supported by the activity of some of the 11 identified types of hemocytes. To study and challenge WBR, it is thus important to have the capacity to isolate, analyze, and manipulate hemolymph in regenerating colonies. Here we present a microtransfusion technique that permits the collection of pure hemocytes, the quantification of their purity, their labeling, and reinjection into colonial tunicates. To exemplify our approach, we present in addition a protocol to analyze the isolated hemocytes using flow cytometry. Our approach is minimally invasive, does not induce lethality, and therefore allows repeated transfusion into exactly the same colony with minimal disruption to the process being studied.

Methods for cell isolation and analysis of the highly regenerative tunicate Polycarpa mytiligera

“…First, we imaged unlabeled dissociated cells from the body wall, and detected autofluorescence in both the green and far-red channels ( Figure 2C ; Methods). These cells appeared granular, in agreement with previous reports of autofluorescence from granular ascidian cells ( Brown et al, 2009 ; Serrato et al, 2022 ). Second, we used a platform combining flow cytometry and imaging (ImageStream; Methods) for detecting autofluorescence in cells in unlabeled body wall samples ( Figure 2D ).…”

“…Isolated cells from ascidians frequently display autofluorescence ( Swalla et al, 1994 ; Brown et al, 2009 ; Serrato et al, 2022 ). To assess the extent of autofluorescence in P. mytiligera , we analyzed unlabeled cells extracted from tissues using flow cytometry and microscopy (Methods).…”

“…Development of a FACS approach for cell purification required autofluorescence analysis, which has been documented in various ascidians ( Swalla et al, 1994 ; Brown et al, 2009 ; Serrato et al, 2022 ). We found that commonly used reagents were applicable to P. mytiligera , following task-specific optimization.…”

“…This is often achieved by using FACS to separate cells based on their morphological properties (e.g., particle size and granularity) and by using functional and genetic fluorescent markers. Cells from marine organisms often have naturally occurring fluorescence (i.e., autofluorescence) ( Swalla et al, 1994 ; Brown et al, 2009 ; Serrato et al, 2022 ). Therefore, application of fluorescent reagents for FACS requires prior assessment of the inherent fluorescent properties of the sample.…”

Methods for cell isolation and analysis of the highly regenerative tunicatePolycarpa mytiligera

“…This is often achieved by using FACS to separate cells based on their morphological properties (e.g., particle size and granularity) and by using functional and genetic fluorescent markers. Cells from marine organisms often have naturally occurring fluorescence (i.e., autofluorescence) [22][23][24] . Therefore, application of fluorescent reagents for FACS requires prior assessment of the inherent fluorescent properties of the sample.…”