Optimized Protocol for Isolation and Culture of Murine Neonatal Primary Lung Fibroblasts

During all the stages of lung development, the lung mesoderm (or mesenchyme) is closely related to the endoderm, and their cross-regulation promotes, controls, and drives all lung developmental processes. Generation of 3D organoids in vitro, RNA assays, and mitochondrial respiration studies are used to analyze lung development and regeneration to better understand the interactions between epithelium and mesenchyme, as well as for the study of redox alterations and the metabolic status of the cells. Moreover, to avoid using immortalized cell lines in these in vitro approaches, standardized murine neonatal primary lung fibroblast isolation techniques are essential. Here, we present an optimized method to isolate, culture, and freeze primary lung fibroblasts from neonatal lungs. Our current method includes step-by-step instructions accompanied by graphical explanations and critical steps.


Introduction
Lung mesoderm proliferation and differentiation must be coordinated with the endoderm through complex signaling networks to give rise to a different subset of differentiated epithelial cells that form the mature lung [1][2][3]. Alterations in this critical interaction can lead to severe anatomical and functional defects in the airway and alveoli, linked to high neonatal mortality in humans [4][5][6]. Hence, analyses of lung mesenchyme biology and function are indispensable to provide knowledge about the role of endogenous lung fibroblasts in several physiological and pathological conditions, as they are implicated in the regenerative potential of the lung epithelium [7][8][9][10]. Understanding the lung mesenchyme impact during lung development, as well as in lung diseases such as idiopathic pulmonary fibrosis (IPF) [11], emphysema [12], or chronic obstructive pulmonary disease (COPD) [13,14] will allow the discovery of new molecular targets used for the development of new therapeutic tools for those diseases.
In this regard, mouse models mimicking these conditions and cell cultures derived from them are useful tools for studies on human illnesses [15]. However, we lack efficient protocols for the isolation and culture of primary fibroblast from newborn lungs, and the existing protocols developed for adult lungs are not suitable [16,17]. In addition, the use of immortalized mouse lung fibroblast cell lines in certain aspects does not represent an appropriate approach as compared with primary cultures.
The aim of the present study is to develop a curated protocol for the isolation and culture of primary lung fibroblasts from newborn mice using an optimized and highly efficient isolation method. The resulting primary cultures are suitable for 2D studies and provide the required support to epithelial lung cells for 3D lung organoids generation.

Experimental Design
The protocol procedures are summarized in Figure 1. Reagents must be prepared using sterile HBSS 1X pH 7.2. Figure 1. Experimental design of the protocol. Collagenase/Dispase (Col/Disp). Red arrows on 6well culture plates represent lung tissue pieces from which lung fibroblasts will crawl-out.

Tissue Collection and Dissociation
(Note: all steps should be performed under aseptic conditions in a biological hood. Hereinafter HBSS 1X will be named as HBSS)

1.
Briefly wash newborn mice skin with 70% Ethanol. Euthanize newborn pups by decapitation. Make a small incision with the scissors from the neck to the chest without reaching the peritoneal cavity, and use forceps to remove the lungs attached to the heart;

Freshly Prepared Solutions (to Be Made Fresh Immediately before the Experiment)
• Collagenase/Dispase ® 1 mg/mL diluted in sterile HBSS 1X. Keep in ice until use.

Tissue Collection and Dissociation
(Note: all steps should be performed under aseptic conditions in a biological hood. Hereinafter HBSS 1X will be named as HBSS) 1. Briefly wash newborn mice skin with 70% Ethanol. Euthanize newborn pups by decapitation. Make a small incision with the scissors from the neck to the chest without reaching the peritoneal cavity, and use forceps to remove the lungs attached to the heart; CRITICAL STEP For multiple pups, euthanize one after another. Do not reach the peritoneal cavity to avoid contaminations; 2. Place the lung lobes attached to the heart into a drop of HBSS in a sterile 100 mm culture plate containing multiple drops of HBSS. Remove the heart and other nonlung tissue, including the trachea and bronchi, and collect the 5 lung lobes individually one by one. Wash out the blood as much as possible by passing the tissue from drop to drop of HBSS at least 3 times ( Figure 2).
CRITICAL STEP For multiple pups, euthanize one after another. Do not reach the peritoneal cavity to avoid contaminations; 2.
Place the lung lobes attached to the heart into a drop of HBSS in a sterile 100 mm culture plate containing multiple drops of HBSS. Remove the heart and other non-lung tissue, including the trachea and bronchi, and collect the 5 lung lobes individually one by one. Wash out the blood as much as possible by passing the tissue from drop to drop of HBSS at least 3 times ( Figure 2).

1.
For the first enzymatic digestion, place the lung lobes into a 1.5 mL Eppendorf tube containing 0.5 mL of 1 mg/mL Collagenase/Dispase ® and mince thoroughly with small surgical scissors. Incubate at 37 • C for 30 min (min) with gentle agitation; 2.
After the incubation, vortex the Eppendorf briefly and centrifuge for 5 min at 1000 × g at room temperature (RT

Freshly Prepared Solutions (to Be Made Fresh Immediately before the Experiment)
• Collagenase/Dispase ® 1 mg/mL diluted in sterile HBSS 1X. Keep in ice until use.

Tissue Collection and Dissociation
(Note: all steps should be performed under aseptic conditions in a biological hood. Hereinafter HBSS 1X will be named as HBSS) 1. Briefly wash newborn mice skin with 70% Ethanol. Euthanize newborn pups by decapitation. Make a small incision with the scissors from the neck to the chest without reaching the peritoneal cavity, and use forceps to remove the lungs attached to the heart; CRITICAL STEP For multiple pups, euthanize one after another. Do not reach the peritoneal cavity to avoid contaminations; 2. Place the lung lobes attached to the heart into a drop of HBSS in a sterile 100 mm culture plate containing multiple drops of HBSS. Remove the heart and other nonlung tissue, including the trachea and bronchi, and collect the 5 lung lobes individually one by one. Wash out the blood as much as possible by passing the tissue from drop to drop of HBSS at least 3 times (Figure 2).

CRITICAL STEP
Do not exceed 20 min digestion time with trypsin, otherwise, the final cell recovery will be extremely low; 5.
Centrifuge for 5 min at 1000 × g at RT. Carefully decant and discard the supernatant.

Enzymatic Digestion
1. For the first enzymatic digestion, place the lung lobes into a 1.5 mL Eppendorf tube containing 0.5 mL of 1 mg/mL Collagenase/Dispase ® and mince thoroughly with small surgical scissors. Incubate at 37 °C for 30 min (min) with gentle agitation; 2. After the incubation, vortex the Eppendorf briefly and centrifuge for 5 min at 1000 × g at room temperature (RT). Carefully decant and discard the supernatant; CRITICAL STEP The digested lung is viscous and does not remain strongly attached at the bottom of the tube. Thus, to avoid losing material, special care is needed when decanting the supernatant; 3. Wash the pellet by adding 1 mL of HBSS and centrifuge for 5 min at 1000 × g. Carefully decant and discard the supernatant; 4. For the second enzymatic digestion, add 0.5 mL of 0.25% Trypsin-EDTA to each Eppendorf and mix thoroughly by pipetting up and down several times. Incubate at 37 °C for 20 min with gentle agitation; CRITICAL STEP Do not exceed 20 min digestion time with trypsin, otherwise, the final cell recovery will be extremely low; 5. Centrifuge for 5 min at 1000 × g at RT. Carefully decant and discard the supernatant.

Red Blood Cell (RBC) Removal
1. Resuspend the pellet in 0.1 mL of RBC at RT and incubate for 1 min. Quickly, neutralize by adding 1.2 mL of 1X PBS; CRITICAL STEP If the erythrocytes are not eliminated, the efficiency will be very low. Do not exceed the lysis time, otherwise, the viability of the fibroblasts will be affected; 2. Centrifuge for 5 min at 1000 × g at RT. Decant and discard supernatant;

Freshly Prepared Solutions (to Be Made Fresh Immediately before the Experiment)
• Collagenase/Dispase ® 1 mg/mL diluted in sterile HBSS 1X. Keep in ice until use.

Tissue Collection and Dissociation
(Note: all steps should be performed under aseptic conditions in a biological ho Hereinafter HBSS 1X will be named as HBSS) 1. Briefly wash newborn mice skin with 70% Ethanol. Euthanize newborn pups by capitation. Make a small incision with the scissors from the neck to the chest with reaching the peritoneal cavity, and use forceps to remove the lungs attached to heart; CRITICAL STEP For multiple pups, euthanize one after another. Do not re the peritoneal cavity to avoid contaminations; 2. Place the lung lobes attached to the heart into a drop of HBSS in a sterile 100 m culture plate containing multiple drops of HBSS. Remove the heart and other n lung tissue, including the trachea and bronchi, and collect the 5 lung lobes indivi ally one by one. Wash out the blood as much as possible by passing the tissue fr drop to drop of HBSS at least 3 times (Figure 2).

CRITICAL STEP
If the erythrocytes are not eliminated, the efficiency will be very low. Do not exceed the lysis time, otherwise, the viability of the fibroblasts will be affected; 2.
Centrifuge for 5 min at 1000 × g at RT. Decant and discard supernatant;

Platting and Freezing
Lung fibroblasts can be maintained as a primary cell culture for up to 6 passages. However, we recommend using a maximum of 4-5 passages.

1.
Resuspend the pellet in 0.5 mL of fibroblast culture media (DMEM supplemented with 10% of FBS, 1% P/S, 0.2% Amphotericin B). Gently pipette up and down to break big aggregates and plate the suspension into a 6-well tissue culture plate; • Antibody solution for imm Triton X-100 in PBS).

Tissue Collection and Dissociat
(Note: all steps should be p Hereinafter HBSS 1X will be nam 1. Briefly wash newborn mice capitation. Make a small inc reaching the peritoneal cav heart; CRITICAL STEP For m the peritoneal cavity to avo 2. Place the lung lobes attach culture plate containing mu lung tissue, including the tr ally one by one. Wash out t drop to drop of HBSS at lea CRITICAL STEP Do not discard tissue pieces as lung fibroblasts will crawl out from those pieces, see Figure 3; 2.
Add 2 mL of fibroblast culture media so the final volume in each well will be about 2.5 mL; 3.
After 24 h, change the media. If erythrocytes are still present in the well, carefully wash with 2 mL of 1X PBS, aspirate PBS and add 2.5 mL of fresh fibroblasts media.
After the first change of media, change it every other day. Split to a 100 mm culture plate when the cells are confluent; 1. Resuspend the pellet in 0.5 mL of fibroblast culture media (DMEM supplemented with 10% of FBS, 1% P/S, 0.2% Amphotericin B). Gently pipette up and down to break big aggregates and plate the suspension into a 6-well tissue culture plate; CRITICAL STEP Do not discard tissue pieces as lung fibroblasts will crawl out from those pieces, see Figure 3;   Place the 3 mL of cells into a 15 mL tube and centrifuge for 5 min at 1200 rpm. Remove the supernatant and resuspend the cell pellet in lung fibroblasts freezing media (10% DMSO, 20% FBS in DMEM). Place cryovial swiftly into ice and freeze up to −80 • C using a cell freezing container. After 24 h, place cryovials into liquid nitrogen.

Immunofluorescence (IF)
To characterize the purity of lung fibroblast isolation, IF against alpha-smooth muscle actin or phalloidin staining can be performed, see Figure 4A,B.
β2-microglobulin is used as a housekeeping gene to have an endogenous control to normalize results. Table 1. Primers used for RT-qPCR assays in lung fibroblasts.

Expected Results and Discussion
Established mouse neonatal cell lines for lung fibroblasts are a useful ready-to-use tool in biology, however, to analyze the mesenchyme from a specific neonatal mouse experimental model, the culture of primary lung fibroblasts is recommended. The currently available protocols for adult lung fibroblast isolation are not suitable for the isolation of primary lung fibroblasts from newborn mice, since performing perfusion to eliminate blood cells is not possible in neonate mice. In this regard, eliminating erythrocytes with RBC lysis buffer is a critical step for a successful primary lung fibroblast isolation, although a quick neutralization is required to avoid losing cell viability. Additionally, the combined enzymatic digestion with Collagenase/Dispase ® and 0.25% Trypsin-EDTA allows a decrease in the overall digestion time (50 min in total), as well as to perform softer digestion of the lung tissue. This avoids excessive cell death and results in a faster primary culture generation.
In this manuscript, we propose an optimized protocol to establish a rapid primary lung fibroblast culture from newborn mice (approximately fully confluent cultures are achieved within 48-72 h after isolation), as compared to those achievable with previously published protocols for adult fibroblast isolation [16,17]. This study also shows that our protocol generates pure lung fibroblast cultures, as demonstrated through immunofluorescence and gene expression assays. Taken together, these results indicate that our combined enzymatic and crawl-out protocol, together with erythrocyte elimination before plating, leads to a high recovery of lung fibroblasts and will be a useful tool for investigating lung mesenchyme biology.  Informed Consent Statement: Not applicable.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.