Thursday, January 29, 2015

Feeding Alex's cells with "Orangina"

Alex’s cells are taking off, they divide and multiply in the culture dish.
Skin biopsy with outgrowth of keratinocytes and fibroblasts



Alex: Where do my cells live now?
Outside of a tissue culture incubator Inside of a tissue culture incubator

Birgitt: Your cells are kept in an incubator at 37C body temperature -like you see on these pictures- and a mixture of gas with 5% CO2. These incubators are located in our tissue culture laboratory. Only trained staff have access to these rooms, which regularly get inspected to ensure proper safety conditions.

Alex:What do you feed my cells? I love pizza and chocolate but that's not what you feed them, right?

Birgitt: Your cells are fed with a cocktail of sugar, protein, and amino acids. It looks like Orangina. Feeding means, you remove the media with a glass pipette under vacuum and then add an ounce or so –depending on the size of the dish- of media with a new sterile pipette to the cells. We change the media every 2-3 days. We also supplement the media with antibiotics to prevent contamination of the cultures with bacteria or fungi.

Alex: I am just a bit competitive. Are my cells competitive too?

Birgitt: Definitely, your cells are growing great. On Day 8, we noticed the first fibroblasts growing out of the biopsy. See the labeled example above to illustrate the outgrowth of keratinocytes and fibroblasts from a skin biopsy.

Below you can see three pictures comparing the growth of your own cells over five days. Fibroblast cells typically double every 24 to 36 hours. The culture dish has some white markings which allow us now to take images from the same area and to follow the dividing cells. You can see how the space between the markings is filling up with cells. Soon this culture dish will be too small and we will have to transfer the cells to a larger dish. In technical jargon we call it “splitting the cells”.

Alex: Thanks so much. I am really amazed that these are my cells.

Wednesday, January 21, 2015

Glossary

Terms are arranged in alphabetical order


2008: 2008 was the year Barack Obama won the presidential elections. Some of his campaign slogans during his election were "change" and "yes we can".

Alive...and in perfect hibernation: A quote from Star Wars: Episode V - The Empire Strikes Back (1980) when Han Solo was frozen in carbonite: Darth Vader "Well, Calrissian? Did he survive?" Lando Calrissian "Yes, he's alive...and in perfect hibernation." http://www.imdb.com/character/ch0000031/quotes

Star Wars Blog


Antibody: Any of a large number of proteins of high molecular weight that are produced normally by specialized B cells after stimulation by an antigen. They act specifically against the antigen in an immune response, that are produced abnormally by some cancer cells, and that typically consist of four subunits. These include two heavy chains and two light chains called immunoglobulin.
http://www.merriam-webster.com/dictionary/antibody 
 
Biological safety cabinet: A laminar flow safety cabinet designed to provide three basic types of protection; Personnel protection from harmful agents inside the cabinet,product protection to avoid contamination of the work, experiment, or process and environmental protection from contaminants contained within the cabinet. http://www.bakerco.com/introduction-biological-safety-cabinets
Watch a video about Sterile Tissue Culture Techniques
Coffee blog


Contact inhibition: Biological process that prevents further division of proliferating cells. (Illustrated Dictionary of Podiatry and Foot Science by Jean Mooney. 2009. Elsevier Limited 24 Feb. 2015)
Growing Out of Clothes Blog

Cryopreservation: The process of cooling and storing cells, tissues, or organs at very low temperatures to maintain their viability. For example, the technology of cooling and storing cells at a temperature below the freezing point ('196' C) permits high rates of survivability of the cells upon thawing. http://www.medicinenet.com/script/main/art.asp?articlekey=7252


Differentiated cell: A cell that has undergone the process of becoming progressively more specialized; developmental process through which cells mature.
Rejuvenation Blog

Embryoid bodies: Spheroid colonies seen in culture produced by the growth of embryonic stem cells in suspension. Embryoid bodies are of mixed cell types, and the distribution and timing of the appearance of specific cell types corresponds to that observed within the embryo. http://www.isscr.org/visitor-types/public/stem-cell-glossary#embryoid

Epitope: The surface portion of an antigen capable of eliciting an immune response and of combining with the antibody produced to counter that response. http://medical-dictionary.thefreedictionary.com/epitope

Feeder cell:Inactivated (non-dividing by radiation or chemically) mouse embryonic fibroblasts (MEFs) provide structural support and release of growth factors for optimal growth of stem cells.

Fibroblasts: Skin fibroblasts are a critical cell type of the skin; they produce and organize the extracellular matrix of the skin and communicate with each other and other cell types. Skin fibroblasts play a crucial role in regulating skin physiology (Sorrell, J.M., and Caplan, A.I. (2004)). Fibroblast heterogeneity: more than skin deep. Journal of cell science 117, 667-675). The fibroblasts have characteristic morphological features of elongated, spindle-like cell bodies, round to oval cell nuclei, and the fibroblasts grow aligned and in bundles when confluent (Vangipuram, M., Ting, D., Kim, S., Diaz, R., and Schule, B. (2013). Skin punch biopsy explant culture for derivation of primary human fibroblasts. Journal of visualized experiments : JoVE, e3779).

The Force: is a metaphysical, spiritual, binding, omnipotent and ubiquitous power that holds enormous importance for both the Jedi and Sith monastic orders. "Well, the Force is what gives a Jedi his power. It's an energy field created by all living things. It surrounds us and penetrates us; it binds the galaxy together. " Obi-Wan Kenobi. http://starwars.wikia.com/wiki/The_Force
Star Wars Blog

The Good, the Bad and the Ugly: Italian Western film directed by Sergio Leone, starring Clint Eastwood, Lee Van Cleef, and Eli Wallach in the title roles respectively.The screenplay was written by Age & Scarpelli, Luciano Vincenzoni and Leone. http://en.wikipedia.org/wiki/The_Good,_the_Bad_and_the_Ugly

Han Solo is a character in the Star Wars movies. Han Solo is portrayed by George Lucas as "a loner who realizes the importance of being part of a group and helping for the common good". He and his co-pilot, Chewbacca, become involved in the Rebel Alliance which opposes the Galactic Empire. During the course of the Star Wars story, he becomes a chief figure in the Alliance and succeeding galactic governments. http://en.wikipedia.org/wiki/Han_Solo
Star Wars Blog

Immunostaining: the staining of a specific substance by using an antibody against it which is complexed with a staining medium (as horseradish peroxidase) http://www.merriam-webster.com/medical/immunostaining 

Human embryonic stem (hES) cells: Embryonic stem cells are derived from embryos that develop from eggs that have been fertilized and then donated for research purposes with informed consent of the donors.("Definition of Differentiation". MedicineNet. MedicineNet, 28 Aug. 2013.

Induced pluripotent stem (iPS) cells: Embryonic stem cell-like cells that were reprogrammed from adult differentiated cells, e.g. skin, blood. The iPS cells have the potential to differentiate into any cell type of the human body. ("What Are Induced Pluripotent Stem Cells? [Stem Cell Information]. National Institute of Health)
Growing Out of Clothes Blog

Karyotype: To arrange and classify the chromosome complement of an organism based on the number, size, shape, and other characteristics of the chromosomes. http://www.biology-online.org/dictionary/Karyotype

Keratinocytes: Keratinocytes are specialized cells that  are predominantly found in the outermost layer of the skin, called epidermis. The primary function of keratinocytes is the formation of a barrier against environmental damage (McGrath JA, Eady RAJ, Pope FM. (2004). "Anatomy and Organization of Human Skin". In Burns T, Breathnach S, Cox N, Griffiths C. Rook's Textbook of Dermatology (7th ed.). Blackwell Publishing. doi:10.1002/9780470750520.ch3. ISBN 978-0-632-06429-8).

Start A Cell Culture Blog

Liquid nitrogen: Liquefied form of nitrogen at an extremely low temperature. Nitrogen boils at −195.79 C (77 K; −320 F) and can cause rapid freezing on contact with tissue. It is used in cryotherapy for treating potentially malignant skin lesions or for the cryopreservation of biological samples such as cells or tissues. http://en.wikipedia.org/wiki/Liquid_nitrogen

Neubauer chamber: The counting chamber or also called hemocytometer is a device used to count cells. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. http://insilico.ehu.es/counting_chamber/neubauer_improved.php

Nuclear or cellular reprogramming: Technology to turn adult differentiated cells into stem cell-like cells using a factors that modulate the expression in the cell nucleus.

Orangina: Orangina is a carbonated citrus beverage made from orange, lemon, mandarin, and grapefruit juices and containing orange pulp. The concept of Orangina originated at a trade fair in France and was first marketed in Algeria by the French Algerian Léon Beton. It is a popular beverage in Europe and to a much lesser extent in North America. http://en.wikipedia.org/wiki/Orangina
Orangina Blog


Philz Coffee: A local California coffee house whose mission is "to better people's day. http://www.philzcoffee.com/
Coffee blog

Pluripotent: Not fixed as to developmental potentialities; capable of differentiating into one of many cell types http://www.merriam-webster.com/medical/pluripotent

Rejuvenation: The process of making young again; illustrated in “Fountain of Youth” by Lucas Cranach the Elder (1472-1553)
Rejuvenation Blog

Skin Biopsy: A procedure in which a sample of skin tissue is removed, processed, and examined ("Skin Biopsy."WebMD.). A skin biopsy can be performed to diagnose unusual skin growth, such as a mole. It is a typically done under local anesthesia with a small pen-like instrument.
Start A Cell Culture Blog

Split (aka Passaging, Subculture): A procedure that enables for further propagation of cell line through the removal of the medium and transfer of cells from a previous culture into fresh growth medium. "Guidelines for Maintaining Cultured Cells". Life Technologies
Growing Out of Clothes Blog

Starbucks: Coffee chain whose motto is "Our mission to inspire and nurture the human spirit – one person, one cup, and one neighborhood at a time." http://www.starbucks.com/

Substantia Nigra: An area in the midbrain which normally includes cells which contain melanin, a byproduct of dopamine synthesis. Parkinson’s disease occurs due to the lack of a dopamine transmitter, causing the degeneration of melanin-containing cells in the Nigra. In deeply affected parkinsonian brains, the band of melanin is absent from the Substantia Nigra. http://www.neuroanatomy.wisc.edu/virtualbrain/BrainStem/20Substantia.html

Trypsin: An enzyme found in the digestive system that hydrolyses proteins. For cell culture, trypsin is used to detach adherent cells from the culture dish during cell harvesting. "Trypsin-EDTA (0.25%)", Stem Cell Technologies.
Growing Out of Clothes Blog

Monday, January 12, 2015

Starting a skin cell culture

Two days ago, Alex donated a small piece of his skin. We are now growing and expanding skin cells from his biopsy and will turn them into stem cells.

Alex: What’s the brown area in the upper left corner of the picture?


Birgitt: This is a piece of your skin how it looks under a microscope. When I took the biopsy two days ago, I used a small instrument that looks like a pen to cut your skin, about the size of a small pea. Later in the lab, I cut the piece into 15 little pieces, which I then transferred to a tissue culture vessel.
We actually have a video showing this process in more detail
http://www.youtube.com/watch?v=pNNL5St3Yhs.


Alex:  Interesting, what else do I see on the picture? There is a rim around the skin tissue with little dots. What are those dots?
Birgitt:  Those dots are cells growing out of the skin biopsy and attach to the bottom of the culture vessel. These cells are called keratinocytes. Soon, we will observe another cell type also known as fibroblasts. These fibroblasts are the cells that we will turn into stem cells.
Alex:  Wow, those are my cells, how exciting! How many skin cells are you growing from me?
Birgitt: In four to six weeks, we will have grown about 20 Million of your skin cells. The journey of your skin cells has begun.