StemCell Therapy

This book is meant for students and professionals who are looking for reference on different areas in this field, to bring a new student or new hire up to speed.

A scientific revolution less than 20 years old that has already changing the foods we eat and react to the environment.

To bring out the best in nature.

Farmers and bakers were the pioneers of the biotech. Remember Grandma's freshly baked bread? How Grandpa kept the seeds of those really big pepper or tomatoes? Your grandparents were practicing biotechnology. Maybe you still do the same, that is the basis of biotechnology.

Defining "Biotechnology"

The application of the principles of engineering and the use of technology in the field of life sciences-bioengineering.

1 The use of living things to make products.2 The study, application and control of a biological processes. 3 The application of any of the above or derivatives thereof, to make or modify products or processes for specifically defined uses.

The use of microorganisms (such as bacteria or yeasts) or biological substances (such as enzymes) to perform specific industrial or manufacturing processes. Applications include the production of certain drugs, synthetic hormones, and bulk foodstuffs, as well as the bioconversion of organic waste and the cleanup of oil spills.

Cloning, genetic manipulation, cell fusion, and mutation.

Modifying the genetic material of organisms directly and with increasing precision, has enabled the transfer of genes between extremely diverse organisms, in combinations unlikely to occur by non-technological means, allowing speedier and more specific results.

Essentially, doing "more and faster" building on what we have known and done for centuries and going beyond.

Life- Defined as:

Products

Good laboratory practice for nonclinical laboratory studies:

http://www.access.gpo.gov/nara/cfr/waisidx_02/21cfr58_02.html

Title 21 Code of Federal Regulations (21 CFR Part 11)Electronic Records; Electronic Signatures

http://www.fda.gov/ora/compliance_ref/part11/

Part 210 - current good manufacturing practice in manufacturing, processing, packing, or holding of drugs; general

Part 211 - current good manufacturing practice for finished pharmaceuticals

http://www.fda.gov/cder/dmpq/cgmpregs.htm

SOP's (Standard Operating Procedures)

Notebook

Documentation for Integrity and traceability

Keys to Successful Biotech products

Record Keeping

requirements

Development / Upstream / Downstream processes

Chemical

Yeast

Fungi

Mammalian Cells

Fermentation?

ExpensiveLabor intensiveOpen EndedTime Consuming

Raw MaterialsBatch to Batch variationsTransportation costsStorage

CompositionGrowth kineticsYieldSeed Bank

Original Stored Cells

Used in actual fermentation

The Biotech Technician must be a person possessing skills with ability to solve problems and meet the customer in such a way that the translations of what is possible can be made clear. They have to maintain a notebook, one that can be read by someone else. Present results in a clear manner, and work with others to meet objectives.

A technician must use the tools of the trade not unlike any other trade, we are farmers but our herd is tiny tiny wildlife. To take care of our herd we must measure certain aspects of their environment.

most accuratemore expensive piece of equipmentStore in bufferCheck for clogging

very coarse measurement of pH

The letters pH stand for "power of hydrogen"

The most abundant element in the universe is hydrogen, which makes up about 3/4 of all matter!

Stronger acids give up more protons, H+ (hydrogen ions); stronger bases give up more OH- (hydroxide ions). Neutral substances have an even balance of H+ and OH-, E.g. Pure (distilled) water.

>7 base -- 7 Neutral -- <7 Acid

Depending on your definition, an acid is a hydrogen ion or proton donator and a base is a hydrogen ion acceptor, hydroxide ion donator, or electron acceptor.

Acids produce H+ ions in aqueous solutions, whereas bases produce OH- ions in aqueous solutions

pH electrode compared to a battery

Store in buffer not H2O

Mercury tubeGood for metals and biologicals and up to 80 degrees C

The common Silver-Silver Chloride reference electrode used with most combination pH electrodes has a Potassium Chloride salt-bridge which is saturated with Silver Chloride.

Works well in most samples, but not in biological samples containing proteins or related materials

Span errorDifference b/w perfect and actual pH Electrode at 25C produces 59.12 mV/pH unit

Offset error

signal @ pH 7.0 @ 25 C is 0 mV

Three point calibration

Calibrate W/I range you going to use

Chemist use buffers to moderate the pH of a reaction.Buffers stabilize a solution at a specific pH value.Resist pH change when small amounts of acid or alkali are added.

KPO4

KPO4 buffer is highly recommended for most P450 assays (microsomal or recombinant enzymes) with the exception of CYP 2C9 and 2A6 where a Tris buffer system is more appropriate.

TRIS buffer

TRIS buffers are used by biochemists to control pH in the physiological range (about 7 to 8 pH) because phosphates cause undesirable side reactions with the biological substances in their test samples.

"Good" buffers

These buffers were well received by the research community because "Good" buffers are nontoxic, easy to purify and their pKa is typically between 6.0 and 8.0, the range at which most biological reactions occur.

The "Good" buffers also feature minimal penetration of membranes, minimal absorbance in the 240-700 nm range and minimal effects due to salt, concentration or temperature.

pKa = dissociation constant

In chemistry and biochemistry, a dissociation constant or an ionization constant is a specific type of equilibrium constant used for dissociation (ionization) reactions.Dissociation in chemistry and biochemistry is a general process in which complexes, molecules, or salts separate or split into smaller molecules, ions, or radicals, usually in a reversible manner. Dissociation is the opposite of association and recombination.

Problems

A gelatinous material derived from certain marine algae.

Two types:

Components required for preparing a minimal agar

LB (Luria-Bertani) Media

contains blood cells from an animal (e.g. a sheep). Most bacteria will grow on this medium

This contains lysed blood cells, and is used for growing fastidious (fussy) respiratory bacteria.

Purpose Mannitol salt agar is both a selective and differential growth medium.

Inhibits Gram+MacConkey

This type of agar is used since it is one of the most forgiving media available - it is hard to contaminate, and E. coli usually grow up as red colonies.

(Almost all spore forming bacteria are Gram-positive, but these cannot grow on MacConkey agar because of the detergent in it (bile salts), and very few Gram-negative bacteria can tolerate either the initial dryness of the plates, or the boiling temperatures needed to make the MacConkey agar. Also, while fungal spores can tolerate the dryness, they cannot tolerate the boiling.)

This is an agar upon which only Gram-negative bacteria can grow

Starch

An agar plate is a sterile Petri dish that contains agar plus nutrients, and is used to culture bacteria or fungi.

contains the antibiotic neomycin.

Used for fungi. It contains gentamicin and has a low pH that will kill most bacteria.

+ Complex+ pH 7.2

Common UV/ VIS spectrophotometers Following is a list of commonly used spectrophotometers: GeneSys 20 HP8452A Diode Array Spectronic 20

Ultraviolet-Visible spectroscopy or Ultraviolet-Visible spectrophotometry (UV/ VIS) involves the spectroscopy of photons (spectrophotometry). It uses light in the visible and adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this region of energy space molecules undergo electronic transitions.

A=elc

There are different types of Sterilization techniques. Some of them are 1. Physical sterilization 2. Chemical sterilization

Under Physical sterilization a) Heatb) Filtration c) Ionising Radiation etc.,In Heat sterilization i. Temperature above 100 Cii. Temperature at 100 Ciii. Temperature below 100 C.

i. Temperature above 100 CThere are two methods involved in it a. Moisture heat sterilizationb. Dry heat sterilization

Using a balanceCalibration / documentation

Gel electrophoresis is a method that separates macromolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties. Researchers can typically control the charge at the top and bottom of the gel. DNA is negatively charged so to run it through the gel, the top would have to be set to - and the bottom to +.

materials

agarose

Agarose is a natural colloid extracted from sea weedIt is very fragile and easily destroyed by handlingAgarose gels have very large "pore" size and are used primarily to separate very large molecules with a molecular mass greater than 200 kDaltonsAgarose gels can be processed faster than polyacrylamide gels, but their resolution is inferior.

Agarose is a linear polysaccharide (average molecular mas about 12,000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose is usually used at concentrations between 1% and 3%. Agarose is a chain of sugar molecules, and is extracted from seaweed.

Perhaps you have seen the terms TBE or TAE.

These are names of two commonly used buffers in electrophoresis.

The "T" stands for Tris, a chemical which helps maintain a consistent pH of the solution.

The "E" stands for EDTA, which itself is another anacronym. EDTA chelates (gobbles up) divalent cations like magnesium. This is important because most nucleases require divalent cations for activity, and you certainly wouldn't want any stray nucleases degrading your sample while it's running through the gel, would you?

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