Air-liquid interface cell culture

Last updated
Pseudostratified epithelium of the trachea ALI cell culture aims to recreate in vitro 2304 Pseudostratified Epithelium.jpg
Pseudostratified epithelium of the trachea ALI cell culture aims to recreate in vitro

Air liquid interface cell culture (ALI) is a method of cell culture by which basal stem cells are grown with their basal surfaces in contact with media, and the top of the cellular layer is exposed to the air. The cells are then lifted and media is changed until the development of a mucociliary phenotype of a pseudostratified epithelium, similar to the tracheal epithelium. [1] [2]

Contents

This method of cell culture aims to be used to study fundamental aspects of the respiratory epithelium, such as cell-to-cell signaling, disease modeling, and respiratory regeneration. [1] [2]

Air-liquid interface cell culture compares to standard cell culture practices by specifically aiming to restore the pseudostratified striation of the respiratory airway in vitro, and aiming to maintain the respiratory airway-niche of (from top to bottom) 1) air, 2) pseudostratified epithelium, and 3) liquid media. Standard cell culture processes are either non-airway specific or revolve around an organ system that requires other means of cellular maintenance in vitro.

Protocol

The protocol for air-liquid interface culture relies on two key steps: isolation of the epithelial cells from an organism, and culture of those cells.

Isolation

Organs with epithelial cells are isolated [1] (dissected) and placed in PBS to clean any impurities and these isolated organs are digested using trypsin. After the digest, ROCK inhibitor (Rho-associated kinase inhibitor) is added to prevent cellular apoptosis of isolated cells. What is left is digested cellular content mixed with tissue; pronase/DNAse is added to clean up dead cellular material as well as any remaining protein material. The resulting material is chopped finely in media and individual isolated cell samples are placed in respectively marked microcentrifuge tubes, and shaken at 37 °C for 30 – 40 minutes. Once the cells have been incubated, they are plated onto transwell membranes for growth.

Culture

Plate cells with SABM (Small Airway Basal Media) to enrich the cells with nutrients for growth and eventual differentiation. [1] Once the cells are plated, they are grown for 3–7 days under careful observation (while changing media each day) until desired confluence is reached.

More recently, a study on In vitro generation of type-II pneumocytes initiated from human CD34(+) stem cells has been demonstrated the air-liquid interface cell culture method precisely. [3]

Uses in scientific study

Air-liquid interface cultures are used in many stem cell studies that aim to recreate a pseudostratified epithelium in vitro . These studies can contribute to new findings on various topics:

Cancer

In modeling cancer and various other diseases, the stem cells in the basal layer of the tracheal epithelium (basal stem cells) were isolated and used in developing 3D organoids that could be used for various studies, including tumor studies. The cell culture method used involves the isolation of cells into culturing with growth factors to grow over time. Once the cells had been grown, they were mixed with Matrigel and cultured to form 3D organoids. [4] [5]

Cell growth/differentiation

In modeling the pseudostratified epithelium in vitro, more cellular studies have been performed in order to determine the nature of the cells – their differentiation pathways, their growth mechanism, and their repair/response mechanism in the state of post-injury. One recent study has shown that differentiated cells in the respiratory epithelium (secretory cells and ciliated cells primarily) can dedifferentiate into their naive status, and become stem-like again. [1] [5]

Adapted protocol: maintenance of stem-like properties in basal stem cells

As an adaptation to the air-liquid interface culture protocol, auxiliary protocols have been developed using the ALI framework to maintain naive stem cell properties for extensive periods of time. [6]

Related Research Articles

<span class="mw-page-title-main">Lung</span> Primary organ of the respiratory system

The lungs are the primary organs of the respiratory system in humans and most other animals, including some snails and a small number of fish. In mammals and most other vertebrates, two lungs are located near the backbone on either side of the heart. Their function in the respiratory system is to extract oxygen from the air and transfer it into the bloodstream, and to release carbon dioxide from the bloodstream into the atmosphere, in a process of gas exchange. The pleurae, which are thin, smooth, and moist, serve to reduce friction between the lungs and chest wall during breathing, allowing for easy and effortless movements of the lungs.

<span class="mw-page-title-main">Pulmonary alveolus</span> Hollow cavity found in the lungs

A pulmonary alveolus, also known as an air sac or air space, is one of millions of hollow, distensible cup-shaped cavities in the lungs where pulmonary gas exchange takes place. Oxygen is exchanged for carbon dioxide at the blood–air barrier between the alveolar air and the pulmonary capillary. Alveoli make up the functional tissue of the mammalian lungs known as the lung parenchyma, which takes up 90 percent of the total lung volume.

<span class="mw-page-title-main">Tissue (biology)</span> Group of cells having similar appearance and performing the same function

In biology, tissue is a historically derived biological organizational level between cells and a complete organ. A tissue is therefore often thought of as an assembly of similar cells and their extracellular matrix from the same embryonic origin that together carry out a specific function. Organs are then formed by the functional grouping together of multiple tissues.

<span class="mw-page-title-main">Gastrulation</span> Stage in embryonic development in which germ layers form

Gastrulation is the stage in the early embryonic development of most animals, during which the blastula, or in mammals the blastocyst is reorganized into a multilayered structure known as the gastrula. Before gastrulation, the embryo is a continuous epithelial sheet of cells; by the end of gastrulation, the embryo has begun differentiation to establish distinct cell lineages, set up the basic axes of the body, and internalized one or more cell types including the prospective gut.

<span class="mw-page-title-main">Respiratory tract</span> Organs involved in transmission of air to and from the point where gases diffuse into tissue

The respiratory tract is the subdivision of the respiratory system involved with the process of respiration in mammals. The respiratory tract is lined with respiratory epithelium as respiratory mucosa.

<span class="mw-page-title-main">Epithelium</span> Tissue lining the surfaces of organs in animals

Epithelium or epithelial tissue is a thin, continuous, protective layer of compactly packed cells with a little intercellular matrix. Epithelial tissues line the outer surfaces of organs and blood vessels throughout the body, as well as the inner surfaces of cavities in many internal organs. An example is the epidermis, the outermost layer of the skin. Epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. These tissues also lack blood or lymph supply. The tissue is supplied by nerves.

<span class="mw-page-title-main">Bronchus</span> Airway in the respiratory tract

A bronchus is a passage or airway in the lower respiratory tract that conducts air into the lungs. The first or primary bronchi to branch from the trachea at the carina are the right main bronchus and the left main bronchus. These are the widest bronchi, and enter the right lung, and the left lung at each hilum. The main bronchi branch into narrower secondary bronchi or lobar bronchi, and these branch into narrower tertiary bronchi or segmental bronchi. Further divisions of the segmental bronchi are known as 4th order, 5th order, and 6th order segmental bronchi, or grouped together as subsegmental bronchi. The bronchi, when too narrow to be supported by cartilage, are known as bronchioles. No gas exchange takes place in the bronchi.

<span class="mw-page-title-main">Mucus</span> Secretion produced by mucous membranes

Mucus is a slippery aqueous secretion produced by, and covering, mucous membranes. It is typically produced from cells found in mucous glands, although it may also originate from mixed glands, which contain both serous and mucous cells. It is a viscous colloid containing inorganic salts, antimicrobial enzymes, immunoglobulins, and glycoproteins such as lactoferrin and mucins, which are produced by goblet cells in the mucous membranes and submucosal glands. Mucus serves to protect epithelial cells in the linings of the respiratory, digestive, and urogenital systems, and structures in the visual and auditory systems from pathogenic fungi, bacteria and viruses. Most of the mucus in the body is produced in the gastrointestinal tract.

<span class="mw-page-title-main">Organoid</span> Miniaturized and simplified version of an organ

An organoid is a miniaturized and simplified version of an organ produced in vitro in three dimensions that mimics the key functional, structural and biological complexity of that organ. They are derived from one or a few cells from a tissue, embryonic stem cells or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities. The technique for growing organoids has rapidly improved since the early 2010s, and The Scientist names it as one of the biggest scientific advancements of 2013. Scientists and engineers use organoids to study development and diseases in the laboratory and industry for drug discovery and development, personalized diagnostics and medicine, gene and cell therapies, tissue engineering and regenerative medicine.

<span class="mw-page-title-main">Respiratory epithelium</span> Mucosa that serves to moisten and protect the airways

Respiratory epithelium, or airway epithelium, is a type of ciliated columnar epithelium found lining most of the respiratory tract as respiratory mucosa, where it serves to moisten and protect the airways. It is not present in the vocal cords of the larynx, or the oropharynx and laryngopharynx, where instead the epithelium is stratified squamous. It also functions as a barrier to potential pathogens and foreign particles, preventing infection and tissue injury by the secretion of mucus and the action of mucociliary clearance.

<span class="mw-page-title-main">Mucociliary clearance</span>

Mucociliary clearance (MCC), mucociliary transport, or the mucociliary escalator, describes the self-clearing mechanism of the airways in the respiratory system. It is one of the two protective processes for the lungs in removing inhaled particles including pathogens before they can reach the delicate tissue of the lungs. The other clearance mechanism is provided by the cough reflex. Mucociliary clearance has a major role in pulmonary hygiene.

Acute inhalation injury may result from frequent and widespread use of household cleaning agents and industrial gases. The airways and lungs receive continuous first-pass exposure to non-toxic and irritant or toxic gases via inhalation. Irritant gases are those that, on inhalation, dissolve in the water of the respiratory tract mucosa and provoke an inflammatory response, usually from the release of acidic or alkaline radicals. Smoke, chlorine, phosgene, sulfur dioxide, hydrogen chloride, hydrogen sulfide, nitrogen dioxide, ozone, and ammonia are common irritants.

<span class="mw-page-title-main">Tracheal cytotoxin</span> Chemical compound

Tracheal cytotoxin (TCT) is a 921 dalton glycopeptide released by Bordetella pertussis, Vibrio fischeri, and Neisseria gonorrhoeae. It is a soluble piece of peptidoglycan (PGN) found in the cell wall of all gram-negative bacteria, but only some bacteria species release TCT due to inability to recycle this piece of anhydromuropeptide.

Cord lining, cord tissue, or umbilical cord lining membrane, is the outermost layer of the umbilical cord. As the umbilical cord itself is an extension of the placenta, the umbilical cord lining membrane is an extension of the amniotic membrane covering the placenta. The umbilical cord lining membrane comprises two layers: the amniotic layer and the sub-amniotic layer. The umbilical cord lining membrane is a rich source of two strains of stem cells (CLSCs): epithelial stem cells (CLECs) and mesenchymal stem cells (CLMCs). Discovered by Singapore-based CellResearch Corporation in 2004, this is the best known source for harvesting human stem cells.

Mitotic cell rounding is a shape change that occurs in most animal cells that undergo mitosis. Cells abandon the spread or elongated shape characteristic of interphase and contract into a spherical morphology during mitosis. The phenomenon is seen both in artificial cultures in vitro and naturally forming tissue in vivo.

<span class="mw-page-title-main">Calu-3</span>

Calu-3 is a human lung cancer cell line commonly used in cancer research and drug development. Calu-3 cells are epithelial and can act as respiratory models in preclinical applications.

Airway basal cells are found deep in the respiratory epithelium, attached to, and lining the basement membrane.

Cultrex Basement Membrane Extract (BME) is the trade name for a extracellular protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and manufactured into a hydrogel by R&D Systems, a brand of Bio-Techne. Similar to Matrigel, this hydrogel is a natural extracellular matrix that mimics the complex extracellular environment within complex tissues. It is used as a general cell culture substrate across a wide variety of research applications.

Intestines-on-a-chip are microfluidic bioengineered 3D-models of the real organ, which better mimic physiological features than conventional 3D intestinal organoid culture. A variety of different intestine-on-a-chip models systems have been developed and refined, all holding their individual strengths and weaknesses and collectively holding great promise to the ultimate goal of establishing these systems as reliable high-throughput platforms for drug testing and personalised medicine. The intestine is a highly complex organ system performing a diverse set of vital tasks, from nutrient digestion and absorption, hormone secretion, and immunological processes to neuronal activity, which makes it particularly challenging to model in vitro.

Jayaraj Rajagopal is an Indian-American physician-scientist. He is the Bernard and Mildred Kayden MGH Research Institute Chair and Professor of Medicine at Harvard Medical School. He founded and serves as the Chief of the Stanbury Physician-Scientist Pathway at the Massachusetts General Hospital Department of Medicine. His laboratory focuses on epithelial biology, lung stem cell biology, regenerative biology, and lung diseases.

References

  1. 1 2 3 4 5 Tata, Purushothama Rao; Mou, Hongmei; Pardo-Saganta, Ana; Zhao, Rui; Prabhu, Mythili; Law, Brandon M.; Vinarsky, Vladimir; Cho, Josalyn L.; Breton, Sylvie (November 2013). "Dedifferentiation of committed epithelial cells into stem cells in vivo". Nature. 503 (7475): 218–223. Bibcode:2013Natur.503..218T. doi:10.1038/nature12777. ISSN   1476-4687. PMC   4035230 . PMID   24196716.
  2. 1 2 "Air-Liquid Interface Culture for Respiratory Research". www.stemcell.com. Retrieved 2018-03-30.
  3. Srikanth, Lokanathan; Venkatesh, Katari; Sunitha, Manne Mudhu; Kumar, Pasupuleti Santhosh; Chandrasekhar, Chodimella; Vengamma, Bhuma; Sarma, Potukuchi Venkata Gurunadha Krishna (February 2016). "In vitro generation of type-II pneumocytes can be initiated in human CD34(+) stem cells". Biotechnology Letters. 38 (2): 237–242. doi:10.1007/s10529-015-1974-2. ISSN   1573-6776. PMID   26475269. S2CID   17083137.
  4. Rock, Jason R.; Onaitis, Mark W.; Rawlins, Emma L.; Lu, Yun; Clark, Cheryl P.; Xue, Yan; Randell, Scott H.; Hogan, Brigid L. M. (2009-08-04). "Basal cells as stem cells of the mouse trachea and human airway epithelium". Proceedings of the National Academy of Sciences. 106 (31): 12771–12775. Bibcode:2009PNAS..10612771R. doi: 10.1073/pnas.0906850106 . ISSN   0027-8424. PMC   2714281 . PMID   19625615.
  5. 1 2 Tata, Purushothama Rao; Rajagopal, Jayaraj (2017-03-01). "Plasticity in the lung: making and breaking cell identity". Development. 144 (5): 755–766. doi:10.1242/dev.143784. ISSN   0950-1991. PMC   5374348 . PMID   28246210.
  6. Mou, Hongmei; Vinarsky, Vladimir; Tata, Purushothama Rao; Brazauskas, Karissa; Choi, Soon H.; Crooke, Adrianne K.; Zhang, Bing; Solomon, George M.; Turner, Brett (2016). "Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells". Cell Stem Cell. 19 (2): 217–231. doi:10.1016/j.stem.2016.05.012. PMC   4975684 . PMID   27320041.