Optimizing Osteoblast Cell Culture for the Study of Dlx Gene Function

1. �Optimizing Osteoblast Cell Culture for the Study of Dlx Gene Function" By: Sean Ghassem-Zadeh

2. Introduction to Dlx Genes � The Dlx gene is responsible for a family of transcription factors which play an important role in areas including the brain, nervous system, and bone formation � The Dlx gene family range from Dlx-1 to Dlx-7 � The Dlx transcription factors have been shown to play an important role in osteoblast development � Specifically, the Dlx-3 gene has been shown to be expressed in the later stages of osteoblast differentation

3. Dlx & Bone Development Research � In order to properly study any life science, quick & efficient means of performing test are needed � emergence of in vitro studies � However, cells within in vitro studies never truly behave like their counterpart cells in an in vivo study � Therefore, quick and efficient means of producing a cell culture that behaves normally is in high value � In the case of Dlx-gene, consistent high yield bone cultures are required in order for proper examination � It is for this reason that there are a plethora of products that help in the cell culture process � One such product is Fetal Bovine Serum (FBS)

4. Fetal Bovine Serum � Serum is defined as the A-cellular portion of the blood of a specific animal. � In the case of Fetal Bovine Serum, fetal calves are bled and the blood is allowed to clot. The remaining liquid remaining after clotting is considered the Fetal Bovine Serum. � For decades, serum from various animals has been added to cell cultures to aid in the attachment and growth of specific cell populations. � Serum contains a spectrum of components ranging from: - Attachment factors (Fibronectin, Laminin, etc.), - Survival/Growth factors (Insulin, IGF-1, etc.), - hormones - vitamins - cofactors.

5. FBS & Its role in osteoblast culture � For decades, scientists have been adding FBS to the media used for cell culture to aid in cell attachment, proliferation, and growth.

6. Mesenchymal Stromal Cell Culture The Calvarial Osteroblast Culture (mCOB) utilizes the parietal lobes of the calvarium of the mice to isolate the �early� osteoblasts which are allowed to proliferate in a unique medium/serum cocktail and later differentiate into mature osteoblasts. The Mesenchymal Stromal Cell Culture (mMSC) utilizes the bone marrow within the long bones of the mice to isolate osteoblast progenitor cells which, after plated will proliferate in a unique medium/serum cocktail and eventually differentiate into mature osteoblasts.

7. Proposed experiment and hypothesis With the need to optimize specific types of cell culture, multiple companies have produced various FBS products that can aid cell culture process. With numerous companies producing multiple lots of FBS, which are obtained from a isolated population of fetal calves, a further investigation is needed to determine the best product for a desired cell culture (in this case calvarial and bone marrow cultures). By screening these different serums along with different lots of the same serum both types of cell cultures, one can determine the most effective product. It is therefore proposed that a specific product list is determined and each serum be tested simultaneously with the same population of cells. In doing so, one can determine the best FBS for the two desired methods of cell culture. It is hypothesized that one will see a slight difference between the various cell cultures as each is slightly different and contain different components. With the difference between the cultures of different serums, one can qualitatively view which serum for its culturing qualities.

8. Serum Test List Control � Hyclone (Lot: ANA18125) Experimental a.) HyClone by Thermo Scientific (Lot: JSL30958) b.)Gibco (Lot: 16000) c.)Gibco (Lot: 26140) d.)Sigma-Aldrich (Lot: 096K0368) e.)Atlantic Biologicals (Lot: B0058) f.)Atlantic Biologicals (Lot: C0078) g.)SAFC Biosciences (Lot: 8A0177) h.)Hyclone (Lot: JRM 28372)

9. mMSC Cell Culture experiment: Materials: a.) a-MEM Media Solution - 10% Fetal Bovine Serum (by volume) � Using 8 experimental and 1 control serum - 1% Pen Strep (by volume) b.) 18 Gauge needle c.) Cell Strainer d.) �-GP Solution e.) Ascorbic Acid Solution (AA) Procedure: Obtaining Bone Marrow Cells a.) Obtain and sacrifice mature mice with gradual CO2 exposure b.) Sterilize mice in a 50/50 solution of ethanol and Wescodyne c.) Cut mouse on ventral side along the abdomen and expose the leg bones by completely removing the skin. d.) Remove foot below knee joint (avoid cutting at joint) e.) Isolate long bones by cutting entire leg at hip joint and place in chilled PBS solution f.) Remove all muscle and connective tissue and place bone back into PBS solution g.) Wash isolated long bones (Femur & Tibia) in PBS solution twice h.) Clean off any remaining connective tissue and insert bones into media solution i.) Place bones in sterile dish and remove both ends of the bone j.) Take up 10mL of media into syringe k.) Insert needle into center cavity of bone and flush out bone marrow while moving needle up and down along bone marrow core. l.) Collect displaced bone marrow and ensure no more than 15mL is used per mouse

10. mMSC Cell Culture experiment continued� Counting/Plating Cells a.) Pipette bone marrow/media solution up & down to ensure continuity of solution b.)Filter out waste and particulate matter through 70�m cell strainer c.) Produce a 1/10 dilution of bone marrow cell solution and view under microscope d.) Calculate total cell count and plate ten million cells per 2.5mL cell culture dish Changing Media a.) On days 4 & 6 change media using original media solution used for bone marrow extraction b.) On day 7 change media to differentiation media which includes: - 8�L �-GP per 1mL of a-MEM media - 2 �L AA per 1mL of a-MEM media c.) Change every other day afterwards until day 21 where maturation has occurred.

11. mCOB Cell Culture Experiment Materials: a.) a-MEM Media Solution - 10% Fetal Bovine Serum (by volume) - 1% Pen Strep (by volume) b.) DMEM Media Solution (Low Glucose) - 10% Fetal Bovine Serum (by volume) - 1% Pen Strep (by volume) c.) �-GP Solution d.) Ascorbic Acid Solution (AA) e.) Digestion Solution (filtered) - 25mg Collagenase P - 32mL PBS - 8mL Trypsin EDTA Procedure: Obtaining Bone Marrow Cells � a.) Obtain and sterilize mice in a 70% ethanol solution b.) Sacrifice newly born mice by decapitation c.)Obtain decapitated calvarium and remove the skin on top of the head d.) Cut out the two parietal lobes of the calvarium and remove the middle sutcher e.) Place removed parietal lobes in PBS solution and rinse 3 times in PBS f.) Aspirate PBS solution and add 10mL of digestion solution to bone g.) Place on rocker for 10 minutes at 37OC h.) Discard first digestion and add 10mL of digestion solution and place back on rocker at same temperature and time length i.) Place second digestion on ice and reintroduce calvarium to digestion solution j.) Repeat steps h & i two more times. k.) Collect and mix three saved digestions and strain through 70�M cell strainer l.) Centrifuge cell solution for 10 minutes at 800xG m.) Re-suspend cells in approximately 10mL of DMEM media solution

12. mCOB Cell Culture Experiment Continued� Counting/Plating Cells a.) Pipette bone cell/media solution up & down to ensure continuity of solution b.) Produce a 1/10 dilution of bone marrow cell solution and view under microscope c.) Calculate total cell count and plate one-hundred thousand cells per 2.5mL cell culture dish Changing Media a.) On days 2 & 4 change media using original media solution used for mCOB isolation b.) On day 6 change media to differentiation media which includes: - 4�L �-GP per 1mL of a-MEM media - 2 �L AA per 1mL of a-MEM media c.) Change every other day afterwards until day 21 where maturation has occurred. �

13. Von Kossa Staining Materials: a.) Formalin Solution (1X) b.) PBS Solution c.)Silver Nitrate Solution Procedure: a.) After cells reach maturation, remove media and wash wells with PBS solution b.) Remove PBS solution and add 1mL of Formalin Solution to perform fixation c.) Allow formalin to sit for 10 minutes and remove d.) Wash wells with water (cells now fixed) e.) Add 1mL of the Silver Nitrate Solution f.) Place in ultraviolet �stratalinker� machine to expose to UV rays g.) Use present time and exposure level and remove from cell h.) Remove silver nitrate and wash cells with water i.) Allow cells to air dry

14. Data & Results

15. Conclusion & Ongoing Trials Contrary to the hypothesis, so far there are no conclusive signs that any one specific serum is more conducive to osteoblast culture than the others. However, due to the length of time required to perform a proper cell culture and the amount of time needed to master the specific techniques, the study is currently on- going and further tests are required to make a conclusive determination of which specific product would optimize the osteoblast cell cultures. No official conclusions can be made at this time however the data thus far suggests that no serum would increase the efficiency of a osteoblast culture for a dlx gene study.

16. Future Experiments Alizarine Red Staining Throughout the course of this experiment, conclusions have been made from qualitative data collected. However, in the future, Alizarine Red staining could offer a quantifiable method for determining the most effective serum for osteoblast cell culture. Alizarine red staining binds with mineralization produced by the osteoblasts after differentiation and afterwards this stain can be removed, collected, and run through a spectrophotometer. This would allow me to quantify the cell culture and allow a more accurate conclusion. Unfortunately due to time constraints, this could not be accomplished.

17. Refrences & Special Thanks *Special thanks to Dr. Lichtler and Dr. Rowe � Li, Haitao, et al. "Expression and function of Dlx genes in the osteoblast lineage." Developmental Biology 316.2 (15 Apr. 2008): 458-470. Academic Search Premier. EBSCO. [Library name], [City], [State abbreviation]. 7 Aug. 2008 <http://search.ebscohost.com/login.aspx?direct=true&db=aph&AN=31564298&site=ehost-live>. � Ghoul-Mazgar, Sonia, et al. "Expression pattern of Dlx3 during cell differentiation in mineralized tissues." BONE 37.6 (Dec. 2005): 799-809. Academic Search Premier. EBSCO. [Library name], [City], [State abbreviation]. 7 Aug. 2008 <http://search.ebscohost.com/login.aspx?direct=true&db=aph&AN=19185374&site=ehost-live>. � �Lamprey Dlx genes and early vertebrate evolutionAdam H. Neidert, Vikrant Virupannavar, Gillian W. Hooker, and James A. Langeland, PNAS 2001;98:1101-1106. � � http://www.nature.com/ejhg/journal/v13/n2/images/5201302f5.jpg