ANIMAL CELL CULTURE

Download ebooks

2010-05-21T18:39:00.000-07:00

2010-05-09T00:28:00.000-07:00

Download this ebooks...
GOOD LABORATORY PRACTICE ebook.
GOOD MANUFACTURING PRACTICE ebook.

Gmp terms

2010-03-14T00:27:00.001-08:00

Acceptance criteria: the product specifications and acceptance/rejection criteria, such as acceptable quality level and unacceptable quality level, with an associated sampling plan, that are necessary for making a decision to accept or reject a lot or batch (or any other convenient subgroups of manufactured units).

Action Levels: microbiological levels in the controlled environment, specified in the standard operating procedures, which when exceeded should trigger an investigation and a corrective action based on the investigation.

Air Sampler: devices or equipment used to sample a measured amount of air in a specified time to determine the particulate or microbiological status of air in the controlled environment.

Airborne Particulate Count (or total particulate count): particles detected are 0.3 um, 0.5 um, and larger. when a number of particles is specified, it is the maximum allowable number of particles per cubic meter of air (or per cubic foot of air).

Airborne viable particulate count (or total airborne aerobic microbial count): when a number of microorganisms is specified, it is the maximum number of colony-forming units (cfu) per cubic meter of air (or per cubic foot of air) that is associated with a cleanliness class of controlled environment based on the airborne particulate count.

Airlock : an enclosed space with two or more doors, which is interposed between two or more rooms, e.g., of differing classes of cleanliness. for the purpose of controlling the airflow between those rooms when they need to be entered. an airlock is designed for and used by either people or goods. (27)

Alert Levels : microbial levels, specified in the standard operating procedures, which when exceeded should result in an investigation to ensure that the process is still within control. alert levels are specific for a given facility and are established on the basis of baseline developed under an environmental monitoring program. these alert levels can be modified depending on the trend analysis done in the monitoring program. alert levels are always lower than action levels.

Aseptic Processing: a mode of processing pharmaceutical and medical products that involves the separate sterilization of the product and of the package (containers/closures or packaging material for medical devices) and the transfer of the product into the container and its closure under microbiologic critically controlled conditions.

Audit: inspection of facilities, functions, or records.

Authorized person: a person responsible for the release of batches of finished product for sale. in certain countries the batch documentation of a batch of finished product must be signed by an authorized person from the production department and the batch test results by an authorized person from the quality control department for batch release.

batch (or lot): a defined quantity of starting material, packaging material or product processed in a single process or series of processes so that it could he expected to be homogeneous. in the case of continuous manufacture, the batch must correspond to a defined fraction of the production, characterized by its intended homogeneity. it may sometimes be necessary to divide a batch into a number of sub-batches, which are later brought together to form a final homogeneous batch.

Batch Number (or lot number): a distinctive combination of numbers and/or letters which specifically identifies a batch on the labels, the batch records, thecertificates of analysis, etc.

Batch Numbering System: standard operating procedure describing the details of the batch numbering.

Batch Records: all documents associated with the manufacture of a batch of bulk product or finished product. they provide a history of each batch of product and of all circumstances pertinent to the quality of the final product. (27) (in reference 27, batch records for production are called “batch processing records” and for the packaging operations “batch packaging records” )

Bioburden: total number of microorganisms detected in or on an article prior to a sterilization treatment.

Biogenerator: a contained system, such as a fermentor, into which biological agents are introduced along with other materials so as to effect their multiplication or there production of other substances by reaction with the other materials. biogenerators are generally fitted with devices for regulation, control, connection, material addition and material withdrawal. (7) (also called a bioreactor) biological agents: microorganisms, including genetically engineered microorganisms, cell cultures and endoparasites, whether pathogenic or not.

Bulk product: any product that has completed all processing stages up to, but not including, final packaging.

Calibration: the set of operations that establish, under specified conditions, the relationship between values indicated by an instrument or system for measuring (especially weighing), recording, and controlling- or the values represented by a material measure, and the corresponding known values of a reference standard. limits for acceptance of the results of measuring should be established.

Cell bank: cell bank system: a cell bank system is a system whereby successive batches of a product are manufactured by culture in cells derived from the same master cell bank [fully characterized for identity and absence of contamination]. a number of containers from the master cell bank are sued to prepare a working cell bank. the cell bank system is validated for a passage level or who/vsq/97.01 105 number of population doublings beyond that achieved during routine production.

Master Cell Bank: a culture of [fully characterized] cells distributed into containers in a single operation, processed together in such a manner as to ensure uniformity and sorted in such a manner as to ensure stability. a master cell bank is usually stored at -70 oc or lower.

Working Cell Bank: a culture of cells derived from the master cell bank intended for use in the preparation of production cell cultures. the working cell bank is usually stored at -70 oc or lower.

Cell Culture: the result from the in-vitro growth isolated from multicellular organisms.

Certification: documented testimaony by qualified authorities that a system qualification, calibration, validation, or revalidation have been performed appropriately and the results are acceptable.

Clean Area: an area with defined environmental control of particulate and microbial contamination, constructed and used in such a way as to reduce the introduction generation, and retention of contaminants within the area.

Clean Room: a room in which the concentration of airborne particles is controlled to meet a specified airborne particulate cleanliness class. in addition, the concentration of microorganisms in the environment is monitored; each cleanliness class defined is also assigned a microbiological level of air, surface, and personnel gear.

Clean/Contained Area: an area constructed and operated in such a manner that will achieve the aims of both a clean area and a contained areas at the same time.

Component: any ingredient intended for use in the manufacture of a drug product, including those that may not appear in such drug product.

Contained Area: an area constructed and operated in such a manner (and equipped with appropriate air handling and filtration) so as to prevent contamination of the external environment by biological agents from within the area.

Containment: the action of confining a biological agent or other entity within a defined space. primary containment: a system of containment which prevents the escape of a biological agent into the immediate working environment. it involves the use of closed containers or safety biological cabinets along with secure operating procedures.

Secondary Containment: a system of containment which prevents the escape of a biological agent into the external environment or into other working areas. it involves the use of rooms with specially designed air handling, the existence of airlocks and/or sterilizers for the exit of materials and secure operating procedures. in many cases it may add to the effectiveness of primary containment.

Control: controls resemble the unknown in composition and are assayed at the same time under the same test conditions by the same method. the results of these tests are used in calculating the mean and standard deviation of the test. controls are used to measure accuracy.

Controlled Environment: any area in an aseptic process system for which airborne particulate and microorganism levels are controlled to specific levels, appropriate to the activities conducted within that environment.

Corrective Action: actions to be performed that are in standard operating procedures and that are triggered by exceeding action levels.

Critical Process: a process that may cause variation in the quality of the pharmaceutical product.

Cross-Contamination: contamination of a starting material. intermediate product, or finished product with another starting material or product during production.

Environmental Isolates: microorganisms that have been isolated from samples from the environmental monitoring program and that represent the microflora of an aseptic processing system.

Environmental Monitoring Program: documented program, implemented through standard operating procedures, that describes in detail the procedures and methods used for monitoring particulates as well as microorganisms in controlled environments (air, surface, personnel gear). the program includes sampling sites, frequency of sampling, and investigative and corrective actions that must be followed if alert or action levels are exceeded. the methodology used for trend analysis is also described.

Finished Product: a product that has undergone all stages of production, including packaging in its final container and labelling.

In-Process Control: checks performed during production in order to monitor and if necessary to adjust the process to ensure that the product conforms to its

Specifications: the control of the environment or equipment may also be regarded as a part of in-process control.

Intermediate Product: partly processed material that must undergo further manufacturing steps before it becomes a bulk product.

Lot: (see terms listed under batch)

Manufacture: all operations of purchase of materials and products, production, quality control, release, storage, shipment of finished products, and the related controls.

Master Formula: a document or set of documents specifying the starting materials with their quantities and the packaging, materials, together with a description of the procedures and precautions required to produce a specified quantity of a finished product as well as the processing instructions, including the inprocess controls.

Master Record: a document or set of documents that serve as a basis for the batch

Documentation (blank batch record).

Material Flow: the flow of material and personnel entering controlled environments should follow a specified and documented pathway that has been chosen to reduce or minimize the potential for microbial contamination of the product/closure/container systems. deviation from the prescribed flow could result in increase in potential for microbial contamination. material/personnel flow can changed, but the consequences of the changes from a microbiological who/vsq/97.01 107 point of view should be assessed by responsible managers and must be authorized and documented.

Media Growth Promotion: procedure that references growth promotion under sterility tests to demonstrate that media used in the microbiological environmental monitoring program, or in media-fill runs, are capable of supporting growth of indicator microorganisms and of environmental isolates from samples obtained through the monitoring program.

Packaging Material: any material including printed material employed in the packaging of a pharmaceutical product excluding any outer packaging used for transportation or shipment. packaging materials are referred to as primary or secondary according to whether or not they are intended to be in direct contact with the product.

Procedures: description of the operations to be carried out, the precautions to be taken and measures to be applied directly or indirectly related to the manufacture of a medicinal product.

Process: set of interrelated resources and activities which transform inputs into outputs.

Processing Instructions: see master formula.

Product Contact Areas: areas and surfaces in a controlled environment that are in direct contact with either products, containers, or closures and the microbiological status of which can result in potential microbial contamination of the product/container/closure system. once identified, these areas should be tested more frequently than non-product-contact areas or surfaces.

Production: all operations involved in the preparation of a pharmaceutical product from receipt of materials, through processing and packaging, to completion of the finished product.

Quality Assurance: a wide ranging concept covering all matters that individually or collectively influence the quality of a product. it is the totality of the arrangements made with the object of ensuring that pharmaceutical products are of the quality required for their intended use.

Quality Control: the part of gmp concerned with sampling, specifications, and testing and with the organizations, documentation, and release procedures which ensure that the necessary and relevant tests are actually carried out and that materials are not released for use, nor products released for sale or supply, until their quality has been judged to be satisfactory.

Quality Management: all activities of the overall management function that determine the quality policy, objectives and responsibilities, and implement them by means such as quality planning, quality control, quality assurance and quality improvement within the quality system.

Quality Policy: overall intentions and direction of an organization with regard to quality, as formally expressed by top management.

Quality: totality of characteristics of an entity that bear on its ability to satisfy stated and implied needs.

Quarantine: the status of starting or packaging materials, intermediates, or bulk or finished products isolated physically or by other effective means while a decision is awaited on their release, rejection, or reprocessing.

Reconciliation: a comparison, making due allowance for normal variation, between the amount of product or materials theoretically produced or used and the amount actually produced or used.

Record: provide a history of each batch of product, including its distribution, and also of all other relevant circumstances pertinent to the quality of the final product.

Reference Standard: any material of known identity and purity or potency. An official reference standard is one obtained from an official source such as bp, or usp, or who. a house reference standard may be obtained by thorough characterization for identity and purity or potency relative to an official reference standard, or by determination of absolute purity by other techniques. depending on the intended use (qualitative or quantitative) and the nature of the assay, a greater or lesser degree of purity is acceptable.

Sampling Plan: a documented plan that describes the procedures and methods for sampling of a controlled environment, identifies the sampling sites, the frequency and number of samples, that analysis of data, and the interpretation of results.

Sampling Sites: documented geographical location, within a controlled environment, where sampling for microbiological evaluation is taken. in general, sampling sites are selected because of their potential for product/container/ closure contacts.

Seed Lot System: a seed lot system is a system according to which successive batches of a product are derived from the same master seed lot at a given passage level. for routine production, a working seed lot is prepared from the master seed lot. the final product is derived from the working seed lot and has not undergone more passages from the master seed lot than the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. the origin and the passage history of the master seed lot and the working seed lot are recorded.

Master Seed Lot: a culture of a micro-organism distributed from a single bulk into containers in a single operation in such a manner as to ensure uniformity, to prevent contamination and to ensure stability. a master seed lot in liquid form is usually stored at or below -70 oc. a freeze-dried master seed lot is stored at a temperature known to ensure stability.

Working Seed Lot: a culture of a micro-organism distributed from the master seed lot and intended for use in production. working seed lots are distributed into containers and stored as described above for master seed lots.

Specification: a document describing in detail the requirements with which the products or materials used or obtained during manufacture have to conform. specifications serve as a basis for quality evaluation.

Standard Operating Procedure (Sop): an authorized written procedure giving instructions for performing operations not necessarily specific to a given product or material but of a more general nature (e.g., equipment operation, maintenance and cleaning; validation; cleaning of premises and environmental control; sampling and inspection). certain sops may be used to supplement product-specific master and batch production documentation.

Sterility: an acceptably high level of probability that a product processed in an aseptic system does not contain viable microorganisms.

Swabs: devices provided that are used to sample irregular as well as regular surfaces for determination of microbial status. the swab, generally composed of a stick with an absorbent extremity, is moistened before sampling and used to sample a specified unit area of a surface. the swab is then rinsed in sterile saline or other suitable menstruum and the contents plated on nutrient agar plates to obtain an estimate of the viable microbial load on that surface.

System: a regulated pattern of interacting activities and techniques that are united to form an organized whole.

Theoretical Yield: the quantity that would be produced at any appropriate phase of manufacture, processing, or packaging of a particular drug product, based upon the quantity of components to be used, in the absence of any loss or error in actual production.

62 Ways To Make 2010 Your Best New Year Yet By Robin Sharma.

2010-03-07T17:42:00.001-08:00

62 Ways To Make 2010 Your Best New Year Yet
By Robin Sharma.

1. Remember that leadership isn’t about your position. It’s about your influence

2. Get fit like a pro athlete

3. Lift people up versus tearing people down

4. Protect your good name. An impeccable reputation takes a lifetime to build. And 60 seconds to lose

5. Surround yourself with positive, ethical people who are committed to excellence

6. Remember that even a 1% daily innovation rate amounts to at least a 100% rate of innovation in 100 days

7. Believe in your dreams (even when others laugh at them)

8. Measure your success, not by your net worth but by your self worth (and how happy you feel)

9. Take an intelligent risk every 24 hours. No try-No Win.

10. Read “Buffett: The Making of an American Capitalist”

11. Watch “Man on Wire”

12. Regardless of your title at work, be a team builder.

13. Remember that business is all about relationships and human connections

14. Say “please” more

15. Say “thank you” more

16. Know your Big 5: the five things that need to happen by the end of this year for you to feel it’s been your best year yet

17. Read your Big 5 every morning while the rest of the world is asleep

18. Read “As You Think”. At least twice this year.

19. Be willing to fail. It’s the price of greatness

20. Focus less on making money and more on creating value

21. Spend less, save more

22. Leave everything you touch better than you found it

23. Be the most positive person in every room you’re in

24. Run your own race

24. Stay true to your deepest values and best ideals

25. Write a handwritten thank you note to a customer/friend/loved one every day

26. When you travel, send love letters to your kids on hotel stationary. In time, they’ll have a rich collection to remember your travels by

27. Read “Atlas Shrugged”

28. Be a problem solver versus a trouble maker

29. Rather than doing many things at mediocrity do just a few things-but at mastery

30. Honor your parents

31. Commit to doing great work-whether anyone notices it or not. It’s one of life’s best sources of happiness.

32. Give more than you receive (another of the truths of happiness)

33. Have your 1/3/5/10/25 years goals recorded on paper and review them weekly

34. Be patient. Slow and steady wins the race. The only reason businesses that went from zero to a billion in a year or two get featured in magazines is because 99% of businesses require a lot more time to win

34. Underpromise and then overdeliver

35. See part of your job as “a developer of people” (whether you work in the boardroom or the mailroom)

36. Wear your heart on your sleeve. When people see you’re real, they’ll fall in love with you

37. Be authentic versus plastic

38. Read “The Alchemist”

39. Remember that life wants you to win. So get out of your own way

40. Consider that behind every fear lives your next level of growth (and power)

41. Eat less food

42. Drink more water

43. Rest when you need to

44. Read “SUCCESS” magazine

45. Write your eulogy and them live your life backwards

46. Demand the best from yourself

47. Remember that the more you go to your limits, the more your limits will expand

48. See everything that happens to you as an opportunity to grow (and therefore, as a precious gift)

49. Be obsessed with learning and self-development

50. Become comfortable alone (you are the only person you get to be with your whole life)

51. Smile. It’s a stunningly effective way to win in business and life

52. Reflect on the shortness of life

53. Be bold when it comes to your dreams but gentle with those you love

54. Remember that success is dangerous because it can kill drive/innovation/passion and going the extra mile. Be successful yet stay hungry

55. Read “The Autobiography of Benjamin Franklin”

56. Be of deep value to this world

57. Own beautiful things but don’t let them own you

58. Use excellent words.

59. Laugh more.

60. Don’t complain, gossip or be negative.

61. Plan as if you’ll live forever but live as if you’ll die tomorrow.

62. Feel free to pass these lessons on to those you want to help.

Glossary/terms

2010-02-21T17:48:00.001-08:00

Glossary/terms

Accreditation: The procedure by which an authoritative body gives formal recognition that a body is competent to carry out specific tasks.

Accuracy: The degree of agreement of a measured value with the actual expected value.

AOAC: Association of Official Analytical Chemists

ASTM: American society for testing and materials.

Calibration: The set of operations that establish, under specified conditions, the relationship between values indicated by a measuring instrument or measuring system, or values represented by material measure and the corresponding values of the measurand.

CITAC: Co-Operation on International Traceability in Analytical Chemistry. A forum for worldwide cooperation collaboration on the mechanisms needed to ensure the validity and comparability o analytical data on a global basis.

cGMP: Current Good Manufacturing Practice.

Code of Federal Regulations (CFR) : Collection of all regulations issued by U.S. government agencies. The individual titles making up the regulations are numbered the same way as the federal laws on the same topic. For example, the Federal Food, Drug, and Cosmetic Act is found in Title 21 of United States Code and the companion regulations implementing the law are found in 21 CFR.

Computer system: A system composed of computer(s), peripheral equipment such as disks, printers and terminals, and the software necessary to make them operate together (ANSI/IEEE Standard 729-1983).

Computerized system: A system that has a computer as a major, integral part.

The system is dependent on the computer software to function.

EPA: Environmental Protection Agency of the United States Government. A regulatory body who develops and enforces all aspects of environmental monitoring. This includes development of analytical methods.

EP: European Pharmacopeia, Official compendium of the

member states of the Council of Europe, which includes

all EC and EFTA countries.

FDA: Food and Drug Administration, U.S. agency, part of the Department of Health and Human Services, responsible for regulating clinical research and approval of marketing

permits for food, drugs, medical devices and cosmetics in the U.S.

GALP: Good Automated Laboratory Practice.

GAMP: Good Automated Manufacturing Practice.

GCP: Good Clinical Practice.

GLP: Good Laboratory Practice

GMP: Good Manufacturing Practice.

ICH: International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use.

ILAC: International Laboratory Accreditation Cooperation. Working for international acceptance of data generated by accredited organizations. Developed the ISO Guide 25.

Installation Qualification (IQ): Documented verification that all key aspects of hardware installation adhere to appropriate codes and approved design intentions and that the recommendations of the manufacturer have been suitably considered.

ISO: International Organization for Standardization. Agency responsible for developing international standards; over 160 technical committees, 650 sub-committees and 1500 working groups; more than 6000 ISO standards published; represents more than 90 countries. Founded in 1947.

5 Tips For How You Can Learn More

2010-02-07T09:46:00.000-08:00

There are a number of reasons why we should be lifelong learners. Continuing education reaps us professional, personal and health benefits that are too numerous to list. However if you do not want to enter another classroom as long as you live then take heart as there are other ways you can be a lifelong learner. Here are five tips for ways to continue your education.

First and foremost is to read. Read for fun and pleasure but also read for information and research. You do not have to fear taking on heavy, dusty tomes filled with multi-syllabic boring language. There is a whole industry of writers who make history and science fun. In fact, you do not even need to read books at all. There are a lot of informative magazines that offer both general and specific information. Check them out at a large book store or your local library before selecting a few to take home. Do not overlook the wealth of information available to you on the internet. There are site and publications devoted to just about any topic you can imagine. Start with a main news site and then begin sifting and searching to find topics that interest you. The best thing about this kind of reading is that you get to pick it -- not a teacher with an agenda you don't understand.

Listening is an often overlooked skill when it comes to learning today. Sure listening in the classroom is important but there are people all around us who have a wealth of knowledge to share if we are only willing to listen. Open your ears and your mind. You might be surprised at what you can learn.

You can also learn by simply observing the life around us. What can you learn about parenting by watching the families around you? You can learn not only what to do but what not to do. What can you learn about financial planning, relationships, and just about everything else you want to know? You can learn from the mistakes of others if you only take the time to be observant.

If you are looking for answers then ask questions. Ask questions of experts as well as information brokers, such as librarians and teachers. You should also ask questions of people like you because they can share their experiences and knowledge.

Finally, the most important part of becoming a lifelong learner is simply pursuing knowledge whether it is for a specific purpose or simply knowledge for the sake of knowledge. It is not important what you decide to study and learn more about and you can change topics as often as you change your shoes if you like. The important thing is to continue opening your mind and filling your brain.

You can becoming a lifelong learner and enjoy continuing education by reading, listening, observing, questioning, and pursuing knowledge. If you take these five steps for continuing education then you will live a smarter and more fulfilling life.

About the author:
Renaissance Woman Deanna Mascle shares more education articles at
http://ezinesbydawggone.info/

Article Source: http://www.Free-Articles-Zone.com

Maintenance and Frozen stock preparation of HEK-293 Cell lines.

2010-01-02T08:06:00.000-08:00

HEK 293 Cell Line
Protocol-at-a-Glance
(PT3494-2)
CLONTECH’s HEK 293 Cell Line is offered for use with the Adeno-XTM Expression Systems. The
general maintenance of HEK 293 cells is described below. Instructions on how to use HEK 293 Cells
to produce infectious recombinant adenovirus (1) are given in all Adeno-XTM Expression System
User Manuals.
Note: Upon receipt, frozen cells should be promptly stored in liquid nitrogen or cultured immediately.
Starting HEK 293 Cell Cultures From Frozen Stocks
Growth Medium: DMEM (or Minimum Essential Medium, a Modification [a-MEM]) supplemented
with 100 units/ml penicillin G sodium, 100 mg/ml streptomycin, 4 mM L-glutamine, and 10% fetal
bovine serum. (Tet System Approved Fetal Bovine Serum is available from CLONTECH, #8630-1.)
1. Thaw 293 cells rapidly by briefly immersing the vial in a 37°C water bath (e.g., 2–3 min with
constant agitation). Upon thawing, immediately wipe the outside of the vial with 70% EtOH,
then transfer the contents of the vial to a 10-cm culture plate or a T25 flask.
2. Add an additional 4 ml of medium to the flask/plate. Gently rock or swirl the flask/plate to
distribute cells evenly over the growth surface. Place the culture in a 37°C, 5% CO2,
humidified incubator.
3. The next day, examine the cells under a microscope. Healthy cells display a flat
morphology and adhere well to the plate. Aspirate the medium and replace with fresh,
prewarmed growth medium.
4. Expand the culture as needed. Cell cultures should be split every 2–4 days, when they
reach 70–80% confluency. As a general rule, HEK 293 cells should not be allowed to
become confluent nor should they be seeded too sparsely. (We suggest plating cells at a
density of 100 cells/mm2.)
Split the cells as follows. Remove the medium and wash the cells once with prewarmed
sterile PBS (containing no Ca2+ or Mg2+). Add 1–2 ml of trypsin-EDTA solution and treat for
1–2 min, or longer, until cells detach. To stop trypsinization, add 5–10 ml of growth medium,
then resuspend the cells gently but thoroughly. Count cells (2), then transfer the desired
number of viable cells to a new culture flask or plate containing an appropriate volume of
growth medium. Gently rock or swirl the plate or flask to evenly distribute the cells.
Preparing Frozen Cultures of HEK 293 Cells
Once you have established HEK 293 cells in culture, we recommend that you prepare frozen stock
from an early passage to ensure a renewable source of cells.
Freezing Medium: Can be purchased from Sigma or prepared (0–20%, a-MEM, 70–90% FBS, and
10% DMSO).
1. Trypsinize cells from the desired number of flasks. (See Step 4 above.)
2. Pool cell suspensions together, count cells, and calculate total viable cell number (2).
3. Centrifuge cells at 125 x g for 10 min. Aspirate the supernatant.
4. Resuspend the pellet at a density of at least 1–2 x 106 cells/ml in freezing medium.
5. Dispense 1-ml aliquots into sterile cryovials.
6. Freeze slowly (1°C per min). If a specialized freezer is not available, you may use
Nalgene’s cryo-containers (#5100) for this purpose (freeze at –70°C overnight). Alternatively,
place vials in a thick-walled styrofoam container at –20°C for 1–2 hr. Transfer to
–70°C overnight. Remove vials from styrofoam container or cryo-containers the following
day and place in liquid nitrogen for storage.
7. Two or more weeks later, confirm the viability of the frozen stocks by starting a fresh culture
from frozen cells as described above.

HEK 293 CELL LINE

2010-01-02T07:55:00.000-08:00

Human Embryonic Kidney 293 cells, also often referred to as HEK 293, 293 cells, or less precisely as HEK cells are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture. HEK 293 cells are very easy to grow and transfect very readily and have been widely-used in cell biology research for many years. They are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy.

Origins of HEK 293 Cells:

HEK 293 cells were generated by transformation of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA in the laboratory of Alex Van der Eb in Leiden, the Netherlands in the early 70s. The human embryonic kidney cells were obtained from a healthy aborted fetus and originally cultured by Van der Eb himself, and the transformation by adenovirus was performed by Frank Graham who published his findings in the late 1970s after he left Leiden for McMaster University in Canada. They are called HEK for human embryonic kidney, while the number 293 comes from Graham's habit of numbering his experiments; the original HEK 293 cell clone was simply the product of his 293rd experiment.

Subsequent analysis has shown that the transformation was brought about by an insert consisting of ~4.5 kilobases from the left arm of the viral genome, which became incorporated into human chromosome 19.

For many years it was assumed that HEK 293 cells were generated by transformation of either a fibroblastic, endothelial or epithelial cell all of which are abundant in kidney. However the fact that the cells originated from cultured kidney cells does not say much about the exact cellular origin of the HEK 293, as embryonic kidney cultures may contain small numbers of almost all cell types of the body. In fact Graham and coworkers more recently provided evidence that HEK 293 cells and several other human cell lines generated by adenovirus transformation of human embryonic kidney cells have many properties of immature neurons, suggesting that the adenovirus was taken up and transformed a neuronal lineage cell in the original kidney culture.

Controversy on the origins of HEK 293 Cells:

As a result of the Shaw et al. study, HEK 293 cells may need to be re-characterized and, as a result, may not be used as an in vitro model for kidney cell function or studies involving kidney cells.

Uses of HEK 293 Cells:

As an experimentally transformed cell line, HEK 293 cells are not a particularly good model for normal cells, cancer cells, or any other kind of cell that is a fundamental object of research. However, they are extremely easy to work with, being straightforward to culture and to transfect, and so can be used in experiments in which the behaviour of the cell itself is not of interest. Typically, these experiments involve transfecting in a gene (or combination of genes) of interest, and then analysing the expressed protein; essentially, the cell is used simply as a test tube with a membrane. The widespread use of this cell line is due to its extreme transfectability by the calcium phosphate method, achieving efficiencies approaching 100% as determined by FACS using a 2XPBS buffer. A lower efficiency might be achievable with an HBS buffer.

An important variant of this cell line is the 293T cell line that contains, in addition, the SV40 Large T-antigen, that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This allows for amplification of transfected plasmids and extended temporal expression of the desired gene products. Note that any similarly domesticated cell line can be used for this sort of work; HeLa, COS and Chinese Hamster Ovary cell are common alternatives.

Examples of such experiments include:
A study of the effects of a drug on sodium channels
Testing of an inducible RNA interference system
Testing of an isoform-selective protein kinase C agonist
Investigation of the interaction between two proteins
Analysis of a nuclear export signal in a protein

A more specific use of HEK cells is in the propagation of adenoviral vectors. Viruses offer an extremely efficient means of delivering genes into cells, since this is what they have evolved to do, and are thus of great use as experimental tools. However, as pathogens, they also present a degree of danger to the experimenter. This danger can be avoided by the use of viruses which lack key genes, and which are thus unable to replicate after entering a cell. In order to propagate such viral vectors, a cell line that expresses the missing genes is required. Since HEK cells express a number of adenoviral genes, they can be used to propagate adenoviral vectors in which these genes (typically, E1 and E3) are deleted, such as AdEasy.

293, and especially 293T, cells are commonly used for the production of lentiviral and retroviral vectors. Various retroviral and lentiviral packaging cell lines are based on these cells.

CHO - Chinese hamster ovary cell line

2010-01-02T07:05:00.000-08:00

Chinese hamster ovary (CHO) cells are a cell line derived from the ovary of the Chinese hamster. They are often used in biological and medical research and commercially in the production of therapeutic proteins. They were introduced in the 1960s and grow as a cultured monolayer. They require the amino acid proline in their culture medium.

CHO cells are used in studies of genetics, toxicity screening, nutrition and gene expression, particularly to express recombinant proteins. Today, CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics.

The use of the Chinese hamster in research began in 1919 where they were used in place of mice for typing pneumococci. They were subsequently found to be excellent vectors for transmission of kala-azar (also known as visceral leishmaniasis), facilitating research in epidemiology.

In 1948, the Chinese hamster was brought to the United States for breeding in research laboratories. In the following years, the Chinese hamster became noteworthy for the cell lines that were derived from its tissues. Having a very low chromosome number (2n=22) for a mammal, the Chinese hamster is an ideal model for radiation cytogenetics and tissue culture.

In 1957, T. Puck obtained a female Chinese hamster from Dr. George Yerganian's laboratory at the Boston Cancer Research Foundation and used it to derive the original Chinese hamster ovary (CHO) cell line. Since then, CHO cells have been a cell line of choice because of their rapid growth and high protein production. They have become the mammalian equivalent of E. coli in research and biotechnology today, especially when long-term, stable gene expression and high yields of proteins are required.

CHO cells do not express the Epidermal growth factor receptor (EGFR), which makes them ideal in the investigation of various EGFR mutations.

SOURCE: WIKIPEDIA

MCF-7 CELL LINE

2010-01-01T06:19:00.001-08:00

MCF-7 is a breast cancer cell line was isolated in 1970 from a 69-year-old Caucasian woman.

In the first of two mastectomies she received the removed tissue was benign, but five years later at her second operation a malignant adenocarcinoma was found. Over the next three years the woman was treated for local recurrences by radiotherapy and hormonotherapy. During the removal of chest wall nodules, a pleural effusion was discovered, from which the MCF-7 cell line was derived in 1970. This cell line retained several characteristics of differentiated mammary epithelium including the ability to process estradiol via cytoplasmic estrogen receptors and the capability of forming domes. This cell line expresses the oncogene Wnt7B.

Originally the karyotype of MCF-7 cells was described of possessing 85 chromosomes around 220 passages later a reduction to 69 had occurred. More recently studies have shown karyotype discrepancies between the MCF-7 cell line from the Michigan Cancer Foundation and the MCF-7 cell line supplied by ATCC (Graham et al. 1986). Comparing the cell lines from the two sources studies concluded that the MCF-7 cells from ATCC could not have been derived from the same individual as the original MCF-7 cell line (Graham et al. 1986).

The primary reference source for the MCF-7 cell line appeared in the Journal of the National Cancer Institute (JNCI) with the citation:

Soule, H. D., Vazquez, J., Long, A., Albert & Brennan, M. (1973). A human cell line from a pleural effusion derived from a breast carcinoma. J. Natl. Cancer Inst. 51, 1409-1413.

Tumor necrosis factor alpha (TNF alpha) inhibits the growth of MCF-7 breast cancer cells. Treatment with anti-estrogens can modulate the secretion of insulin-like growth factor binding protein's.

PIK3CA helical mutations were identified in MCF-7, but with low AKT activation.

OSMOLARITY

2009-06-28T17:42:00.000-07:00

BACK TO HOME

OSMOLARITY

Definition of osmolarity:

The concentration of osmotically active particles expressed in terms of osmoles of solute per litre of solution.

Osmolarity is determined by osmometer. An osmometer is a device for measuring the osmotic strength of a solution, colloid, or compound.

What is osmole?

In chemistry, the osmole (osmol) is a non-SI unit of measurement that defines the number of moles of a chemical compound that contribute to a solution's osmotic pressure. The term comes from the phenomenon of osmosis, and is typically used for osmotically-active solutions. For example, a solution of 1 mol/L NaCl corresponds to an osmolarity of 2 osmol/L. The NaCl salt particle dissociates fully in water to become two separate particles: an Na+ ion and a Cl- ion. Therefore, each mole of NaCl becomes two osmoles in solution. Similarly, a solution of 1 mol/L CaCl2, gives a solution of 3 osmol/L (Ca2+ and 2 Cl-).

A strict definition of an osmole is the amount of osmotically active particles that when dissolved in 22.4 L of solvent at 0 degrees celsius exerts an osmotic pressure of 1 atmosphere. This is equivalent to the observation that 1 mole of a perfect gas occupying a volume of 22.4 L exerts a pressure of 1 atmosphere.

1 osmole = 1 mole of osmotically active particles.

mosmol: A unit of osmotic pressure equal to one thousandth of osmole or osmotic pressure of 0.001 molar solution of a substace that does not dissociate.

Formula for Calculating Osmolarity

If one does not have an osmometer, the osmolarity of medium can be increased by a calculated amount using the following method:

1. Use 0.0292 g of NaCl per liter to raise the osmolality 1 mOsmole.

2. Therefore, if you use a 5 M NaCl stock solution (theoretically 10,000 mOsmole), 1 ml/liter will raise the osmolality 10 mOsmole.

Example: Medium A has an osmolarity of 300 mOsmole according to the manufacturer's information.

One wishes to study the effect of increasing osmolarity in the range of 300¨C400 mOsmole.

Experimental Conditions: medium A at 300, 325, 350, 375, and 400 mOsmole.

1. Control 300 mOsmole = Medium A with no additions

2. 325 mOsmole = 10 ml medium A + 25 μl sterile 5 M NaCl solution

3. 350 mOsmole = 10 ml medium A + 50 μl sterile 5 M NaCl solution

4. 375 mOsmole = 10 ml medium A + 75 μl sterile 5 M NaCl solution

5. 400 mOsmole = 10 ml medium A + 100 μl sterile 5 M NaCl solution

BACK TO HOME

cell culture

2009-06-13T20:02:00.000-07:00
Cell culture techniques are very important to all biotechnologist. this site is going to add more information regarding animal cell culture