Efficient isolation of interstitial fibroblasts directly from mouse kidneys or indirectly after ex vivo expansion

Abstract

Renal interstitial fibroblasts are responsible for producing the erythroid growth factor Epo and the vasopressor renin in addition to kidney fibrosis, in which they are transformed into myofibroblasts. Therefore, analyses of fibroblasts may elucidate the complex mechanisms of kidney diseases. However, the fragility of these cells makes their isolation for in vitro analyses and ex vivo cultivation difficult. We have overcome these difficulties by mildly dissociating mouse kidneys and coculturing fibroblasts with other kidney cells in semisolid medium. For complete details on the use and execution of this protocol, please refer to Sato et al. (2019a) and Miyauchi et al. (2021).

Keywords: Cell culture; Cell isolation; Flow Cytometry/Mass Cytometry; Tissue Engineering.

Graphical abstract

Figure 1

Overview of the protocol to isolate interstitial fibroblasts from a mouse kidney A mouse kidney is minced and digested with Collagenase II before being serially filtered with 70-μm and 30-μm cell strainers. The flow-through (single-cell suspension) from the 30-μm cell strainer is directly utilized for cell sorting to isolate primary renal interstitial fibroblasts. Remnants (kidney pieces) on the 70-μm strainer are collected and cultivated in semisolid medium for 1–2 weeks to expand renal interstitial fibroblasts, followed by cell sorting.

Figure 2

Preparation of kidney pieces (A) Mouse kidneys before (left) and after (right) renal medulla removal. (B) Kidneys are minced with scissors (left) until they become a smooth paste (right). (C) Minced kidneys are washed with PBS in a C-tube three times. The left and right panels show the first and third washes, respectively. Clear supernatant (PBS) is removed after 1 min of standing. (D) Kidney pieces are collected on a 70-μm strainer. The remnants on the 70-μm strainer are collected by washing the strainer, which is face down, with DMEM containing FBS.

Figure 3

Isolation of REP cells from REP cell-reporter mice before or after cultivation in semisolid medium (A) The expression of tdTomato red fluorescent protein and green fluorescent protein (GFP) in kidney cell suspensions of anemic REP cell-reporter mice before (left, step 19) and after (right, step 37) primary culture for 6 days is analyzed by flow cytometry. In the reporter mice, tdTomato is expressed by Cre-mediated recombination of the Rosa26-loxP-STOP-loxp-tdTomato modified gene in cells that have ever expressed the EpoCre transgene (Yamazaki et al., 2013). The anemic condition of the reporter mouse enhances the expression of GFP from the endogenous Epo allele (left), in which GFP cDNA is integrated, because Epo gene expression is induced by anemic hypoxia (Obara et al., 2008). During the primary culture, GFP expression disappears (right). The percentage of tdTomato⁺ cells (red boxes) increases from 0.1% to 0.6% after primary culture. (B) tdTomato fluorescence is detectable in cell pellets (arrows) of sorted tdTomato⁺ fibroblasts (right) but not in those of tdTomato^– cells (left). (C) tdTomato expression in a kidney piece from the REP cell-reporter mouse. (D) tdTomato expression in cells primarily cultured for 6 days in semisolid medium.