Rabbit Primary Lung Microvascular Endothelial Cells

Description

Establishing a consistent rabbit lung microvascular endothelial cell culture requires clearly defined tissue origin, passage, endothelial characterization, and post-thaw handling conditions. Variations in coating, medium preparation, storage, or recovery procedures can affect primary cell attachment and short-term expansion.

Rabbit Primary Lung Microvascular Endothelial Cells, Catalog No. N-6011, are isolated from the lung tissue of New Zealand White rabbits. The cells are grown in gelatin-precoated tissue culture flasks with Cell Biologics' Complete Growth Medium and cryopreserved at passage 3.

Each frozen vial contains 0.5 × 10⁶ cells/mL. Endothelial identity is characterized by fluorescence-labeled acetylated low-density lipoprotein uptake, or DiI-Ac-LDL uptake, a functional marker for endothelial cells.

The cells test negative for bacteria, yeast, fungi, and mycoplasma. Under the specified culture conditions, these primary rabbit lung endothelial cells can be expanded for 3–5 passages using a recommended split ratio of 1:2. Repeated freezing and thawing are not recommended.

Specifications

Medium verification: The supplied N-6011 datasheet lists H1168. Confirm the medium catalog number before ordering or publishing this page.

Features

• Primary microvascular endothelial cells isolated from New Zealand White rabbit lung tissue
• Harvested and cryopreserved at passage 3
• Each frozen vial contains 0.5 × 10⁶ cells/mL
• Characterized by functional DiI-Ac-LDL uptake
• Tested negative for bacteria, yeast, fungi, and mycoplasma
• Expandable for 3–5 passages under the specified culture conditions
• Recommended 1:2 split ratio for primary cell expansion
• Maintained in gelatin-precoated culture vessels
• Frozen and live-cell delivery formats are documented

Quality Control

Cell Culture Workflow

Culture Preparation

• Perform all cell culture procedures in a biosafety cabinet.
• Use aseptic techniques to prevent microbial contamination.
• Sterile-filter culture media, supplements, and reagents through a 0.2 µm filter.
• Prewarm only the amount of culture medium needed for immediate use to 37°C.
• Use 5 mL of medium for a T25 flask and 8–10 mL for a T75 flask when recovering cells, changing medium, or splitting cells.
• Avoid repeatedly warming complete culture medium.

Gelatin Coating

• Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution, Catalog No. CB6950, for 2 minutes.
• Aspirate the excess coating solution before seeding cells.

Recovery from a Cryopreserved Vial

1. Thaw the cells rapidly in a 37°C water bath for less than 1 minute or until only a few ice pieces remain.
2. Immediately remove the vial and disinfect its exterior with 70% ethanol.
3. Transfer the cells into a sterile 15 mL conical tube containing 5 mL of prewarmed Cell Biologics culture medium.
4. Rinse the vial with an additional 0.5–1 mL of medium to recover remaining cells.
5. Centrifuge at 200 × g for 5 minutes to remove residual DMSO.
6. Carefully aspirate the supernatant and resuspend the cell pellet in 5 mL of growth medium.
7. Seed the resuspended cells into a gelatin-precoated T25 flask.
8. Incubate at 37°C with 5% CO₂ for 12–16 hours.
9. Change the medium on the second day and every 24 hours thereafter.
10. Use 5 mL per T25 flask or 10 mL per T75 flask for daily medium changes.
11. Check the cells daily for confluence and appropriate morphology.

Primary Cell Expansion

1. Remove the culture medium and wash the cells twice with room-temperature 1X PBS without calcium or magnesium.
2. Add prewarmed 0.25% Trypsin-EDTA Solution, Catalog No. CB6914.
3. Use 0.5–1 mL for a T25 flask or 2 mL for a T75 flask.
4. Incubate for 3–5 minutes and monitor cell detachment.
5. When the cells detach, immediately add 5–10 mL of culture medium containing fetal bovine serum to neutralize the trypsin.
6. Gently pipette the cells up and down several times.
7. Seed the cells into fresh gelatin-precoated flasks or plates.
8. Return the culture vessels to a humidified 37°C, 5% CO₂ incubator.
9. Use the recommended split ratio of 1:2.
10. Change the medium on the second day and every 24 hours thereafter.

Live-Cell Arrival Handling

• Live cells may be supplied in T25 or T75 flasks.
• Upon arrival, remove the sticker from the filter cap.
• Place the flask, containing 6–20 mL of existing culture medium, in a 37°C, 5% CO₂ incubator for 1 hour.
• Replace the existing medium with the desired Cell Biologics culture medium prewarmed to 37°C.
• Subculture at a 1:2 ratio when the cells reach 90% confluency.
• Cells may be frozen at 95–100% confluency for future use.

Preparation for Cell-Based Experiments

• Once the cells reach confluence, they may be allowed to overgrow for 24 hours before cell-based assays, staining, FACS analysis, or other designed experiments.
• Continue daily medium changes and wash the cells twice with calcium- and magnesium-free 1X PBS between medium changes.
• Before an experiment, the protocol recommends filtering cells through a 40 µm cell strainer to remove cell clumps and most dead or floating large cells.

Cryopreservation

• Use Freezing Medium, Catalog No. CB6916, containing 10% DMSO, 50% FBS, and 40% culture medium.
• Add 1 mL of freezing medium per vial and rapidly resuspend the cell pellet.
• Place the vials in a Nalgene “Mr. Frosty” freezing container filled with 100% isopropanol.
• Freeze at -80°C for 6–12 hours as a temporary controlled-freezing step.
• Transfer the vials to a liquid nitrogen tank for long-term storage.

Applications

Rabbit Primary Lung Microvascular Endothelial Cells provide a defined, lung-derived primary endothelial cell source for research workflows requiring New Zealand White rabbit microvascular endothelial cells. The supplied documents support their use in primary cell culture, short-term expansion, staining, FACS analysis, and other investigator-designed cell-based experiments performed under the specified culture conditions.

• In vitro culture of primary rabbit lung microvascular endothelial cells
• Research workflows requiring rabbit pulmonary microvascular endothelial cells
• Functional endothelial cell characterization using DiI-Ac-LDL uptake
• Short-term primary endothelial cell expansion for 3–5 passages
• Cell-based assays performed after the culture reaches confluence
• Staining and FACS analysis performed according to the supplied protocol
• Investigator-designed experiments using rabbit lung-derived endothelial cells

Specific disease models, marker expression profiles, and assay performance are not stated in the supplied N-6011 documents and should be independently evaluated for the intended experimental workflow.

Storage and Handling

• Cryopreserved cells are shipped with dry ice overnight.
• Immediately transfer frozen cells to liquid nitrogen storage at -180°C upon arrival.
• Do not use a -20°C or -80°C freezer for routine or long-term product storage.
• The -80°C step described in the freezing protocol is limited to a temporary 6–12 hour controlled-freezing period before transfer to liquid nitrogen.
• Repeated freezing and thawing are not recommended.
• Treat all animal cells as potential pathogens because no testing procedure can completely guarantee the absence of infectious agents.

Recommended Culture Materials

Documents

N-6011 Product Datasheet

Primary Cell Thawing, Culture, Expansion and Freezing Protocol

Frequently Asked Questions

What are Rabbit Primary Lung Microvascular Endothelial Cells?

They are primary microvascular endothelial cells isolated from the lung tissue of New Zealand White rabbits. N-6011 cells are grown in gelatin-precoated culture flasks and cryopreserved at passage 3.

Are rabbit lung and rabbit pulmonary microvascular endothelial cells the same product type?

“Lung” and “pulmonary” are commonly used to describe the same tissue location. The formal name documented for N-6011 is Rabbit Primary Lung Microvascular Endothelial Cells.

How many cells are supplied in a frozen vial?

The product datasheet states that each frozen vial contains 0.5 × 10⁶ cells/mL.

At what passage are N-6011 cells cryopreserved?

The cells are harvested and cryopreserved at passage 3.

How are the endothelial cells characterized?

They are characterized by fluorescence-labeled acetylated low-density lipoprotein uptake, or DiI-Ac-LDL uptake, a functional marker for endothelial cells.

What quality control testing is documented?

The product datasheet states that the cells test negative for bacteria, yeast, fungi, and mycoplasma.

How many passages can the cells be expanded?

Under the specified Cell Biologics culture conditions, the cells can be expanded for 3–5 passages using a recommended split ratio of 1:2.

What culture conditions are recommended?

The protocol specifies a humidified incubator maintained at 37°C with 5% CO₂. Culture vessels must be precoated with gelatin-based coating solution before seeding.

Which coating solution is recommended?

The protocol specifies Gelatin-Based Coating Solution, Catalog No. CB6950. Coat the flask or culture dish for 2 minutes and aspirate the excess solution before seeding cells.

How frequently should the medium be changed?

Change the medium on the second day after seeding and every 24 hours thereafter. The protocol specifies 5 mL for a T25 flask and 10 mL for a T75 flask during daily medium changes.

How should cryopreserved rabbit lung endothelial cells be thawed?

Rapidly thaw the vial in a 37°C water bath for less than 1 minute, disinfect the vial, transfer the cells into prewarmed medium, and centrifuge at 200 × g for 5 minutes to remove residual DMSO. Resuspend the pellet and seed the cells into a gelatin-precoated T25 flask.

Can N-6011 cells be stored in a -80°C freezer after arrival?

No. Transfer the frozen cells to liquid nitrogen at -180°C immediately upon arrival. The -80°C step in the freezing protocol is only a temporary 6–12 hour controlled-freezing step before transfer to liquid nitrogen.

Is live-cell shipment available?

Yes. The product datasheet states that live-cell shipment is available on request. The supplied protocol includes arrival instructions for live cells delivered in T25 or T75 flasks.

Can these primary endothelial cells be repeatedly frozen and thawed?

No. Repeated freezing and thawing are not recommended.

Are N-6011 cells intended for clinical or diagnostic use?

No. These cells are for research use only and are not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures.