Artificial Blood The blood of the future : Artificial Blood The blood of the future By
Anjali Thakkar
COSMOS 2007
Cluster 1
Mentor: Professor Paul Feldstein
The History of Artificial Blood: The History of Artificial Blood Milk was one of the first substances used as a blood substitute in order to treat patients with Asiatic cholera. (Squires 1).
After several patients died by receiving milk transfusions, other substances were discovered as potentials (Squires 1):
Salt or saline solutions: used primarily as a plasma volume expander, rather than as artificial blood
Hemoglobin isolated from red blood cells
Animal plasma could be used as a substitute for human blood
However, since many of the materials in animal plasma are toxic to humans, this posed a problem to using it as a substitute
The problem of not having a workable substitute led to Ringer’s Solution…
Ringer’s Solution: Ringer’s Solution Created by the physiologist Sydney Ringer in 1882
Ringer’s solution is a saline solution which is able to prolong the life of cells in the body
Solution contains sodium chloride, potassium chloride, calcium chloride, and sodium bicarbonate: designed in the concentrations found in the human body to keep the heart pumping even outside of the body
This solution resembles blood serum
Often used to culture animal cells (Encyclopedia Britannica)
Blood Grouping: Karl Landsteiner: Blood Grouping: Karl Landsteiner http://nobelprize.org/nobel_prizes/medicine/laureates/1930/landsteiner.jpg Karl Landsteiner was a world-renowned immunologist and pathologist in the 1900s.
contributed to the world of medicine in the fields of anatomy, histology, and immunology
1909: Landsteiner classified human blood into four different groups: A, B, AB, and O
Recognized the agglutinins in the blood
This has become the basis for modern blood typing today
received the Nobel Prize for Physiology or Medicine in 1930 (Nobel Foundation)
Artificial Blood vs. Blood Substitutes: Artificial Blood vs. Blood Substitutes
Ideal Characteristics of Artificial Blood: Ideal Characteristics of Artificial Blood Safe to use
Compatible in the human body
Able to transport and release oxygen where needed
Storable and durable for longer time periods
Is free of pathogens and toxins which would produce an immune system response in the human body (Squires 3).
Problems Currently Associated with Artificial Blood and Blood Substitutes: Problems Currently Associated with Artificial Blood and Blood Substitutes Bodily immune systems may sometimes react negatively to the foreign blood that is inserted into the body (Goorha et al 46).
Trauma/Shock Patients
Since these patients are often frequent recipients of blood substitutes or plasma during surgery, it becomes challenging to understand which types of blood substitutes have affected which problem in the patient’s body (Winslow 2).
Currently no real working source of artificial blood exists that can perform the multifarious tasks of real human blood cells (Goorha et al 46).
Types of Blood Substitutes Perfluorocarbons (PFC) emulsions: Types of Blood Substitutes Perfluorocarbons (PFC) emulsions Perfluorocarbons are derived from a group of hydrocarbons in which the hydrogen atoms are replaced by fluorine atoms.
PFCs are chemically inert due to the strength of the carbon-fluorine bonds
Used to create artificial blood during surgeries
Process of production:
Water, salts, and phospholipids surfactant are added and emulsified through high-pressure homogenization
Purified through high temperatures of steam (Goorha et al 47-48).
Common PFCs:
Perfluorodecalin
Perflubron
Types of Blood Substitutes Perfluorocarbons (PFC) emulsions: Types of Blood Substitutes Perfluorocarbons (PFC) emulsions Oxygenation:
Saturation occurs with PFCs passively as oxygen molecules “dissolve into molecular cavities within droplets of the liquid” ("Types of Blood Substitutes”)
Thus, the oxygenation of PFCs is directly related to the partial pressure of oxygen which is in contact with the PFC (See figure below)
"Types of Blood Substitutes." eurobloodsubstitutes.com. 2007. Euro Blood Substitutes. 31 Jul 2007
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Types of Blood Substitutes Perfluorocarbons (PFC) emulsions: Types of Blood Substitutes Perfluorocarbons (PFC) emulsions Structure:
Perfluorocarbon core
Surrounded by a phospholipid surfactant (Werlin et al)
A surfactant is “a substance that reduces the surface tension of the liquid in which it is dissolved” (Brown, useful definitions)
http://biomed.brown.edu/Courses/BI108/BI108_2005_Groups/10/webpages/PFClink.htm
Types of Blood Substitutes Perfluorocarbons (PFC) emulsions: Types of Blood Substitutes Perfluorocarbons (PFC) emulsions
Types of Blood Substitutes Hemoglobin-based Oxygen Carriers (HBOCs): Types of Blood Substitutes Hemoglobin-based Oxygen Carriers (HBOCs) Hemoglobin-based Oxygen Carriers were created as a mechanism to mimic the oxygen-carrying role of hemoglobin in the body, while still reducing the need for real human hemoglobin.
Hemoglobin is a tetramer with two alpha and two beta polypeptide chains; each is bound to an iron heme group which successively binds to an oxygen molecule
Hemoglobin’s heme bond allows it to have a higher affinity for oxygen, thus making it an excellent source of blood substitutes. (Goorha et al 46).
Types of Blood Substitutes Hemoglobin-based Oxygen Carriers (HBOCs): Types of Blood Substitutes Hemoglobin-based Oxygen Carriers (HBOCs) Currently, experimentation is occurring to develop vesicles which carry the Hemoglobin before inserting it into the body as an HBOC
There are several advantages to HBOC carrier vesicles
Prevents the denaturation of the Hemoglobin
The vesicles themselves are made of purified Hb and lipids, making them compatible with the human immune system
Current Setbacks with Hb Vesicle Technology
Materials such as nylon, gelatin, and gum arabic have been tried but the human immune system, specifically the reticuloendothelial system, removed the vesicle promptly as a natural bodily response.
Later, the theory of phospholipid vessels was used to create a vesicle using phospholipids
Researchers are trying to develop a way to create liposome sacks to carry the HBOCs (Goorha et al)
Types of Blood Substitutes: Types of Blood Substitutes Goorha, Brig, and Maj Deb. "Artificial Blood." MJAFI 59(2003): 45-49.
Types of Blood Substitutes: Types of Blood Substitutes Goorha, Brig, and Maj Deb. "Artificial Blood." MJAFI 59(2003): 45-49. This figure displays the different forms of blood substitutes
that can be used to simulate the actions of a red blood cell
Researchable Question: Researchable Question Can we make a blood substitute using Biotechnological techniques?
In order to create a blood substitute that would effectively transport oxygen throughout the body, the hemoglobin gene would have to be inserted in a plasmid vector and multiplied in a specific medium.
The hemoglobin gene would be inserted into the plasmid to carry the cloned gene into E. Coli cells
This would allow for expression of large amounts of the hemoglobin protein
Escherichia Coli P678-54: Escherichia Coli P678-54 The particular strain of the bacteria E. Coli P678-54 has the unique property of dividing abnormally – it forms a mother cell and a daughter cell (Marszalone et al 1219)
The mother cell is similar to a regular cell in that it contains chromosomal DNA. However, it contains ALL of the cell’s DNA
The daughter cell is achromosomal. It is smaller, and contains no chromosomal DNA.
The daughter cell is often referred to as a “mini cell”
Because E. Coli P678-54 has the property of dividing abnormally, it would be ideal if used as a Gene Delivery Vehicle, or GDV (Cohen et al 64).
Since E. Coli P678-54 has a tendency to divide into mother cells as well as mini cells, these mini cells could be ideal for the creation of hemoglobin-based oxygen carriers (Giacalone et al 1).
Method of HBOC production using E. Coli P678-54 Mini cells: Method of HBOC production using E. Coli P678-54 Mini cells In order to produce a hemoglobin-based oxygen carrier using E. Coli P678-54 mini cells, the hemoglobin gene must be inserted into the E. Coli bacterium.
The general process to insert hemoglobin into E. Coli cells uses several techniques of biotechnology:
PCR – to assemble the human hemoglobin gene
Western Blotting – to ensure that the substance being produced really is the Hemoglobin protein
Inoculation – to grow the E. Coli cells after transformation
Electroporation – to open the E. Coli cell pores and allow the plasmid vectors containing the Hb gene to enter
Blue/White Screening – to test whether or not the E. Coli cells have been successfully transformed with the Hb gene
Gene Transformation – to incorporate the Hb gene into E. Coli DNA through plasmid vectors
Enzyme digestion – to properly insert the Hb gene into the plasmid vector containing the lacZ indicator gene
Experimental Design - Materials: Experimental Design - Materials Agar gel plate with Ampicillin antibiotic
Plasmids with the lacZ indicator gene
Micropipettes
E. Coli P678-54 cells
Human DNA
Primers (to make complete PCR products of the human hemoglobin gene)
Experimental Design – Methods Hemoglobin Gene Preparation: Experimental Design – Methods Hemoglobin Gene Preparation Preparation of Hemoglobin
Obtain and purify hemoglobin DNA
Use primers to cut hemoglobin DNA and remove introns
Use modification methods to ensure that the cell free hemoglobin does not break down and lose its properties
This newly assembled hemoglobin DNA can be used as a PCR product
PCR Product:
PCR buffer
dNTP
Hemoglobin primer mix
Sterile water
PCR (Polymerase Chain Reaction)
Run the PCR product through a PCR machine
Inserting hemoglobin into plasmid vectors
Using a plasmid vector with the lacZ indicator gene, we can insert the hemoglobin DNA along with primers for both ends so that the hemoglobin can enter the plasmid
This plasmid now contains the recombinant hemoglobin DNA
Experimental Design – Methods Transformation of E. Coli Cells: Experimental Design – Methods Transformation of E. Coli Cells The plasmid vectors are inserted into E. Coli P678-54
Insert the plasmid vectors into a solution containing E. Coli P678-54
Place the solution through “electroporation”: This allows the plasmid vectors to enter the E. Coli cells by opening up pores in the E. Coli
Blue/White Screening
Once the plasmid vectors have been inserted into the E. Coli cells, spread the transformed cells onto an agar plate with the Ampicillin antibiotic
White colonies: represent colonies which have hemoglobin properly inserted into the plasmids the hemoglobin plasmid has successfully broken the lacZ gene region
Blue colonies: represent colonies which haven’t been properly transformed: the lacZ gene displays the blue color; thus, colonies that show the blue color have the lacZ region still intact, meaning that the hemoglobin gene has not been incorporated into the region
Experimental Design – Methods Mini Cell Separation: Experimental Design – Methods Mini Cell Separation Separation of Mini Cells from normal cells
Suspend the solution by microcentrifuging it
Mini cells will be suspended higher than the normal cells as they are less dense
Pipette out the supernatant above the mini cells
Pipette the mini cells without disturbing the normal E. Coli cells
Experimental Design – Methods Testing for Oxygen: Experimental Design – Methods Testing for Oxygen Testing transformed E. Coli minicells for oxygen content
a simple way to test the transformed mini cells for oxygen binding to hemoglobin would be to check whether the cells are red or blue
Oxygenated blood = red
Deoxygenated blood = blue
Check the Hemoglobin Saturation percentage (Wikipedia)
This is a sigmoidal (non-linear) function which measures the partial pressure of oxygen
In a healthy human being, 98.5% of the oxygen is bound to Hemoglobin, thus making it oxygenated
1.5% of the oxygen is bound to other liquids, making it inaccessible
How to check
Infrared absorption between oxygenated and deoxygenated blood varies highly, thus allowing hospitals and other clinics to differentiate between oxygenated blood and deoxygenated blood
Experimental Design – Methods Hemoglobin Modification Methods: Experimental Design – Methods Hemoglobin Modification Methods Ensuring the stability of hemoglobin
When hemoglobin is left outside a cell, it has the tendency to break into its individual parts, instead of remaining as an entire hemoglobin protein (“Types of Blood Substitutes”)
Methods have been discovered to retain the stability of hemoglobin including:
"Types of Blood Substitutes." eurobloodsubstitutes.com. 2007. Euro Blood Substitutes. 31 Jul 2007
Further Research: Further Research Since hemoglobin has been inserted into E. Coli cells, the body’s immune system will most likely develop antibodies to combat the foreign E. Coli cells that are inserted
Option 1: the mini cells could be packaged in a liposome to trick the immune system (Li 3761).
Option 2: we could find the gene in E. Coli that stimulates the production of antibodies and use gene silencing techniques to turn the gene off
Works Cited: Works Cited Cohen, Amikam and W. D. Fisher. "DNA Isolated from Escherichia Coli Minicells Mated with F+ Cells." Proceedings of the National Academy of Sciences of the United States of America 61 (1968) 61-68. 17/07/2007.
Giacalone , Matthew, and Gentile, Angela, and Lovitt, Brian, and Xu, Tong, and Sabbadini, Roger, and Surbur, Mark. "The Use of Bacterial Minicells to transfer Plasmid DNA to eukaryotic cells." Cellular Microbiology 8 (2006) 1624-1633. 16/07/2007 .
Goorha, Brig, and Maj Deb. "Artificial Blood." MJAFI 59(2003): 45-49.
Li, Shuliang. "Liposome-encapsulated actin–hemoglobin (LEAcHb) artificial blood substitutes." Biomaterials 26June 2005 3759-3769. .
Works Cited: Works Cited Marszalek, Piotr and Tian Tsong. "Cell Fission and Formation of Mini Cell Bodies by High Frequency Alternating Electric Field." Biophysical Journal 68 April 1995 1218-1221. 17/07/2007.
Nobel Foundation, "Karl Landsteiner." Nobelprize.org. 2007. Nobel Foundation. 31 Jul 2007 .
“Ringer's solution." Encyclopedia Britannica. 2007. Encyclopedia Britannica Online. 23 July 2007 .
Squires, Jerry E. "Artificial Blood." Science 295February 2002 1002-1005. 17/07/2007 .
"Types of Blood Substitutes." eurobloodsubstitutes.com. 2007. Euro Blood Substitutes. 31 Jul 2007 .
Werlin, Evan. "Current Synthetic Blood Products." biomed.brown.edu. April 2005. Brown University. 31 Jul 2007 http://biomed.brown.edu/Courses/BI108/BI108_2005_Groups/10/webpages/PFClink.htm
Wikipedia, "Blood." Wikipedia.com. July 2007. Wikipedia. 31 Jul 2007 .