The document describes a proposed research study to develop an ex vivo 3D model of chronic lymphocytic leukemia (CLL) using polyhydroxybutyrate (PHB) scaffolds. The study involves: 1) Producing PHB through bacterial fermentation to fabricate scaffolds for culturing CLL cells. 2) Fabricating and characterizing PHB, commercial PHB, and polyurethane scaffolds. 3) Evaluating the scaffolds' ability to support cell seeding and long-term proliferation of four CLL cell lines over several weeks.

1. “ Development of an ex vivo 3-D model of chronic lymphocytic leukemia (CLL)” SAIFUL IRWAN ZUBAIRI SUPERVISOR: Dr. Sakis Mantalaris CO-SUPERVISOR: Dr. Nicki Panoskaltsis

2. Brief research introduction: No one could ever wonder the complex renewable feedstocks - e.g.: tapioca hydrolysates, whey, food scraps from our daily consumption and pulp fiber sludge - ground breaking materials - T.E.R.M (Koutinas et al., 2006). Recently, scientist has discovered a few natural polymers - complex renewable sources - produced - aliphatic group (e.g.: PHA and PHB) - used effectively - support niches structure known as scaffolds. Natural scaffolds - deliver self renewal hematopoietic stem cells (HSCs) - transplanting - the scaffolds + HSCs into the leukemic patient’s bone marrow - adverse effects.

3. Continue: The main interest - natural polymers - polyhydroxybutyrate (PHB). Poly  -hydroxybutyric acid (PHB) is the most extensively studied PHA - produced in nature in the presence of excess carbon by bacteria as storage granules - provide food and energy - during its limited food sources (Pfeffer, 1992; Salehizadeh and Van Loosdrecht, 2004). PHB has properties similar to petroleum derived synthetic plastics - polypropylene (PP) - completely biodegradable in the environment (Anshuman et al., 2006). Negative gram bacterial ( Ralstonia eutropha a.k.a Alcaligenes euthropus NCIMB 11599) - produce - polyhydroxybutyrate (PHB) - via batch and fed-batch fermentation process.

4. Aim and hypothesis 1 (MPhil level): (1) To synthesize natural polyesters (PHB) from the pure commercial nutrient media (high in carbon and nitrogen sources) by using batch and fed-batch bacterial fermentation process. It is hypothesized that by using the pure commercial nutrient media - the high yield of PHB (mg) can be obtained - purity up to 60% - regardless - batch or fed-batch fermentation process - an excellent benchmark/standard as if the REAL renewable feedstocks are used as a nutrient supplement.

5. Aim and hypothesis 2 (MPhil level): (2) To fabricate scaffolds from the naturally synthesized PHB (bacterial fermentation) and commercialized natural origin PHB (Sigma-Aldrich, 99%) and compared it with the non-biodegradable polyurethane (PU) from the previous studies. It is hypothesized that by using a 3-D culture system that resembled the highly porous structure of the bone marrow - a good depiction of the original leukemic bone marrow architecture can be reproduced . Leukemic cell culture could be maintained with its original genetic features - independent from cytokine addition - HOPEFULLY - natural CLL growth could be studied in its natural condition ex vivo .

6. Aim and hypothesis 3 (MPhil level): (3) To evaluate the efficacy of all cell seeded scaffolds by checking its cell seeding efficiency and long term proliferation. This investigation is based on the hypothesis that 3-D growth design for CLL proliferation will imitate the in vivo marrow environment more closely than 2-D culture - resulting in an enhanced in vitro culture without the need to add exogenous cytokines. Two natural and one synthetic based polymeric scaffolds are selected and tested as suitable materials for CLL seeding and growth. These scaffolds will have interrelated pores to allow cell penetration and multiply - to mimic the bone marrow structure.

7. What is leukemia in relation with BM? Bone marrow - complex 3-D tissue - hematopoiesis is regulated by the intercellular microenvironments niches. The dysregulation of this niche, either in structure or function - can contribute in the pathogenesis (step-by-step development of disease) of a certain disease - e.g.: Leukemia ( Lowenberg et al ., 1999). Leukemia = a cancer of the blood or bone marrow - is characterized by an abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). The latest statistics - incurable blood tumors which are known as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) - the main common blood tumors - kill approximately 1.4 millions peoples in United States for the past 20 years ( Leukemia & Lymphoma Society, 2008).

8. Continue: Leukemia - clinically and pathologically subdivided into several large groups. The first division is between its acute and chronic forms. To be more specific, the diseases are subdivided according to which kind of blood cell is affected. In lymphoblastic or lymphocytic leukemias - the cancerous change takes place in a type of marrow cell that normally goes on to - lymphocytes, which are infection-fighting immune system cells. In myeloid or myelogenous leukemias - the cancerous change takes place in a type of marrow cell that normally goes on to - red blood cells, some other types of white cells, and platelets.

9. Combining these two classifications provides a total of four main categories: Definition in relation with rate of the cell growth: Acute Leukemia = an overgrowth of very immature blood cells - progressing quickly - this is a life threatening situation - low in mature blood cells - anemia, infection & bleeding to death. Chronic Leukemia = an overgrowth of mature blood cells – progressing slowly

10. What is chronic lymphocytic leukemia (CLL)? Chronic lymphocytic leukemia - a type of leukemia or cancer of the white blood cells (lymphocytes). CLL affects a particular lymphocyte - e.g.: the B cell - originates in the bone marrow, develops in the lymph nodes, and normally fights infection. In CLL, the DNA of a B cell is damaged - it can't fight infection - it grows out of control and crowds out in the healthy blood cells that can fight infection. CLL is a disease of ADULTS . Most people newly diagnosed with CLL are over the age 50 (>75%) - majority are men. In the United States during 2007 - it is estimated there are 15,340 new cases diagnosed and 4,500 deaths, but because of prolonged survival - due to medicine breakthrough - many more people are living with CLL. Early CLL is not treated. Late CLL is treated with chemotherapy and monoclonal antibodies . Survival varies from 5 years to more than 25 years.

11. What is Polyhydroxylbutyrate (PHB)? Polyhydroxybutyrate (PHB) belongs to and is the most common member of a broader class of polyesters - polyhydroxyalkanoates (PHA). It is an intracellular accumulated by a wide range of microorganisms - carbon and energy reserve material in response to an environmental stress - e.g.: nutrient limitation. Among all microbes - Ralstonia eutrophus a.k.a Alcaligenes eutrophus - produced - the highest % dry weight of biomass - 96 % (w/w) - purity of PHB - up to 60 % (w/w). Over 80 different PHAs have been classified - each varies in mechanical properties. The difference within the polymers depends on the R side chain . In particular, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biocompatibility properties with human tissues make it appealing for the use in biomedical applications .

12. Ralstonia eutrophus a.k.a Alcaligenes eutrophus -ve gram bacteria. Non-spore forming bacillus . Growth at T = 30 o C (optimal) - environment that contain toxic heavy metal (mM). Synthesize PHB - as a way to store LIPIDS - excess carbon is present but limited in nitrogen and phosphate.

13. Metabolic Pathway for PHB accumulation Consists of three major steps which is involved in 3 types of enzymes (Verlinden, 2007): 1. 3-ketothiolase - produces acetoacetyl-CoA by joining two molecules of acetyl-CoA 2. Acetoacetyl-CoA reductase - promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA. 3. PHA synthase - polymerizes the 3-hydroxybutyryl-CoA to form PHB. Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria. R-chain side PHB - consist - combination of 20,000 monomers

14. Why PHB but not other classes of polyester (e.g.: PHA, PHV, PHH & etc.)? Most of the polyesters (aliphatic & aromatic) are water soluble and moisture sensitive - hydrolysis process (degradation induced by water). But for PHB - the only polyester - H 2 O insoluble. **Although the cells adhere & grow better on hydrophilic surface scaffolds (Lee et al., 1998; Van et al., 1985) - PHB can be treated to be slightly hydrophilic by using the lipases and NaOH treatment ( Deng et al., 2002)** Relatively resistant to hydrolytic degradation. Non-toxic materials. Shows good O 2 permeability. Soluble in chloroform and other chlorinated hydrocarbons (e.g.: methyl chloride, dichloromethane).

15. Research Approach (MPhil Level)

16. (1) Acquiring the polyhydroxybutryrate (PHB) - Bacterial batch and fed-batch fermentation process: The batch and fed-batch fermentation process are conducted in triplicate on 2 different occasions/treatment (N = 6). There are 4 dependant variables that needs to be observed which are as follows: (a) total glucose consumed (mg); (b) microbial biomass concentration (mg/ml); (c) purified total dry weight (mg); and (d) yield of PHB (mg/mg). The results are expressed as mean  standard deviation (mean  SD). Significantly different results (between the treatments) are evaluated by using one-way analysis of variance (ANOVA) with a level of p<0.05 or p<0.01 depending on the measurement that considered significant .

17. Continue: A level of p<0.05 or p<0.01 is considered as significant data (significantly different with the other treatments). The fermentation media are formulated by mixing the pure glucose (30 mg/ml) + protein hydrolysates (30 mg/ml) + yeast extracts (5 - 10 mg/ml) + sterile tap water (to dissolve all nutrients). The nutrient is supplemented with mineral - which contained 1.0 mg/ml KH 2 PO 4 + 0.005 mg/ml CaCl 2 + 0.1 ml trace element solution. The fed-batch fermentation process : runs up to 70 - 100 hrs ( Kim et al., 1994) - the amount of pure glucose consumed (mg) & microbial biomass concentration (mg/ml) - measure - interval time of 10 hrs. The batch fermentations process : the amount of pure glucose consumed (30 mg/ml) - observed - 1 hr interval despite the fact that the batch process usually end up earlier than the fed-batch process. The estimated time is between 35 - 50 hrs (Koutinas et al., 2006) - the time could be vary depending on the kinetics of the fermentation process.

18. (2) Production of scaffolds: Fabrication and characterization 3 types of scaffolds are fabricated in the lab by using T.I.P.S. 1 scaffold: naturally synthesized PHB - R. eutropha fermentation process. 2 other scaffolds: (a) commercialized natural origin PHB (Sigma-Aldrich); (b) polyurethane (PU) from the previous studies. Polyurethane (PU) - is chosen - can significantly mimic the in vivo micro environment of BM - allowing cells to proliferate profoundly. Both are meant for comparison only. The experiments - triplicate for each of the scaffolds. The total numbers of experiment: 9 (N = 9). 4 dependant variables need to be observed which are as follows: (a) porosity (%); (b) mass of the approximate 5x5x5 mm 3 cubes (g); (c) scaffold volume (V s : ml); (d) pores volume (V p : ml). Dependant variables for (a), (c) and (d) - Pycnometer The results are expressed as mean  standard deviation (mean  SD).

19. Type of polymeric materials which are used for the fabrication of scaffolds and comparison purposes:

20. (3) Product testing (scaffolds + cells): Evaluating the cell proliferation 4 cell line cultures for each of 3 different scaffolds are evaluating on 12 different occasions (4 cell lines  3 different scaffolds), in triplicate on each occasions. The total numbers of treatment are 36 (N = 36). The scaffolds are evaluated in terms of seeding efficiency (%) - which is measured 24 h after seeding - and cellular proliferation using the MTS assays at different time intervals of: 48 hr, 1, 2, 3, and 4 weeks. Once the best scaffolds suitable for CLL expansion is identified - long-term culture tests are performed on the selected ones (up to 8 weeks). Finally, the results obtained are compared with PU - a conclusion of the most suitable scaffold can be identified. The results data are expressed as mean  standard deviation (mean  SD). Significantly different of the results (between the treatments) are evaluated by using one-way analysis of variance (ANOVA) with a level of p<0.05 or p<0.01 depending on the measure considered significant . A level of p<0.05 or p<0.01 is considered as significant data (significantly different with the other treatments). Data are analyzed by linear regression using SPSS version 17.0 (SPSS Inc. Ca).

21. Cell seeding and proliferating observation of 4 different types of human’s CLL cell lines into 3 different types of polymeric materials

22. “ THANK YOU FOR YOUR KIND ATTENTION”

23. Metabolic Pathway: Continue